CN103517902B - Methods and compositions for treating beta-thalassemia and sickle-cell anemia - Google Patents

Abstract

The present invention provides compounds that induce gamma globulin expression (e.g., compounds of formula I-a) and pharmaceutical compositions thereof. The compounds may have beneficial therapeutic effects. The compounds and compositions described herein are useful for treating hemoglobinopathies, such as beta-thalassemia and sickle cell anemia.

Description

Methods and compositions for treating beta-thalassemia and sickle-cell anemia

Cross-references to related applications

This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application U.S.S.N. 61/444,436, filed February 18, 2011, which is incorporated by reference in its entirety.

Background of the invention

The human hemoglobin molecule consists of a protein heterotetramer (two α-like (α-like) hemoglobin chains and two β-like (β-like) hemoglobin chains) and four non-protein heme groups . The α-like and β-like hemoglobin gene clusters are located on different chromosomes, and the expression of the different hemoglobin genes within the two gene clusters is temporally controlled during development. Genetic defects (such as deletions or mutations) within these hemoglobin loci lead to abnormal synthesis of hemoglobin and thus lead to hemopathies.

Beta-thalassemia and sickle cell anemia are the two most common hemopathies, together affecting about 4.5% of the world's population. Beta-thalassemia is caused by deletions or mutations within the beta-like hemoglobin gene cluster that reduce the synthesis of beta-hemoglobin chains in adults. Severe deficiency or absence of β-globulin chains can lead to an imbalance in the expression of α-globulin chains in adults, and excessive α-globulin chains can in turn precipitate and damage the red blood cell membranes, eventually inducing red blood cells in early erythrocytic blasts. During rapid apoptosis (also known as beta-thalassemia major or Cooley's anemia). Individuals with β-thalassemia major become fully anemic within 6 to 9 months of life, the time when the conversion of hemoglobin from HbF (α2/γ2) to HbA (α2/β2) is complete.

Sickle cell anemia is caused by a point mutation (Glu to Val) at position 6 of the beta hemoglobin chain. Patients with sickle cell anemia are characterized by the presence of sickle hemoglobin HbS (α2/ ^βS2 ). Mutated adult beta hemoglobin chains promote HbS polymerization under hypoxic conditions, which distorts red blood cells into their characteristic sickle shape. Sickle cell disease is mainly caused by hemolysis, as the malformed sickle cells are destroyed in the spleen within 10-20 days. Due to the high risk of early death, the life expectancy of patients with sickle cell anemia has been reported to be shortened to an average of 42-48 years. Because normal adult hemoglobin production is impaired, patients with beta-thalassemia major and sickle cell anemia require regular blood transfusions to replenish functional HbA in order to survive. However, continuous blood transfusions are associated with high costs (over $100 million per year in the US alone) and a high risk of iron overload, which often leads to death.

In beta-thalassemia and sickle cell anemia, it has been reported that an increase in the expression of HbF contributes to the improvement of the clinical symptoms of the underlying disease. In patients with β-thalassemia major, the synthesis of fetal γ-globulin chains can be increased to form HbF and thereby balance the excess α-globulin chains, thus regulating severe anemia in patients. In addition, increased gamma globulin chains also prevent HbS formation, and the presence of HbF directly inhibits HbS aggregation in sickle cell patients.

Therefore, pharmacological induction of HbF in hemepathic patients may serve as a potentially useful therapeutic strategy. To date, several chemotherapeutic agents have been shown to stimulate fetal hemoglobin production, such as trichostatin A (histone deacetylase inhibitor), apicidin (histone deacetylase enzyme inhibitor), 5'-aza-cytidine (DNA methyltransferase inhibitor), hydroxyurea (ribonucleotide reductase inhibitor), butyrate and other short-chain fatty acids. However, most of these HbF inducers display variable efficacy from individual to individual, low specificity of hemoglobin gene induction, and high toxicity of irreversible apoptosis. Among these drugs, hydroxyurea is the first drug approved by the US FDA for the treatment of hemoglobinopathy. Unfortunately, approximately 25% of users respond poorly or non-responsively to hydroxyurea therapy. In addition, there are potential side effects of myelosuppression and reproductive toxicity, which raise therapeutic concerns about the use of hydroxyurea in patients.

In view of this, compounds that induce the expression of endogenous embryonic/fetal hemoglobin chains for the treatment of β-thalassemia major and sickle cell anemia have received great clinical attention.

Summary of the invention

The present invention provides compositions and methods for treating beta-thalassemia and sickle cell anemia by inducing endogenous embryonic/fetal hemoglobin chains, including gamma hemoglobin gene induction in red blood cells. The methods of the invention comprise administering to a subject a pharmaceutical composition comprising a therapeutically effective amount of a compound of the invention effective to treat, delay or prevent adverse effects of beta-thalassemia or sickle cell anemia. In certain embodiments, a therapeutically effective amount of a compound described herein is effective to induce expression of embryonic/fetal hemoglobin chains.

In some embodiments, pharmaceutical compositions of compounds for the treatment of beta-thalassemia or sickle cell anemia by inducing endogenous embryonic/fetal hemoglobin chains are provided. Provided compositions comprise an effective amount of a compound described herein and a pharmaceutically acceptable excipient. In other embodiments, compounds are provided that have the property of inducing the expression of endogenous embryonic/fetal hemoglobin chains in cells of the erythroid lineage that may be present in culture in a mammalian host or as an in vitro model .

In one aspect, the invention provides pharmaceutical compositions comprising a compound of formula (I-a):

or a pharmaceutically acceptable salt, solvate or hydrate thereof, wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ^6a , R ^6b and n are as defined herein. In some embodiments, the pharmaceutical composition provided herein can be used to treat β-thalassemia or sickle cell anemia.

In another aspect, the invention provides a method of inducing gamma globulin comprising contacting a cell with a compound or composition provided herein. In certain embodiments, the contacting step is performed in vitro. In other embodiments, the step of contacting is performed in vivo. In certain embodiments, the present invention provides methods of treating beta-thalassemia comprising administering to a patient in need thereof an effective amount of a compound or composition provided herein. In other embodiments, the present invention provides methods of treating sickle cell anemia comprising administering to a patient in need thereof an effective amount of a compound or composition provided herein.

In another aspect, the invention provides methods of identifying inducers of gamma globulin expression. In certain embodiments, the method comprises incubating a test compound with MEL cells containing a human gamma hemoglobin promoter, a beta hemoglobin promoter, and a dual fluorescent reporter plastid; and measuring the fluorescence compared to background signal strength. In certain embodiments, the present invention provides MEL cells comprising a human gamma globulin promoter, a beta hemoglobin promoter, and a dual fluorescent reporter plastid.

definition

The practice of the present invention will employ, unless otherwise indicated, techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the skill of the art. Such techniques are fully explained in, for example, "Molecular Cloning: A Laboratory Manual", 2nd Edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (ed. M.J. Gait, 1984); "Animal Cell Culture" (R.I. Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (D.M.Weir & C.C. Blackwell, ed.); "Gene Transfer Vectors for Mammalian Cells" (J.M.Miller & M.P. Calos, ed. 1987); "Current Protocols in Molecular Biology" (F.M. Ausubel et al., eds., 1987); "PCR: The Polymerase Chain Reaction", (Mullis et al., 1994); and "Current Protocols in Immunology" (J.E. Coligan et al., eds., 1991).

As used throughout, "modulate" means to increase or decrease a specified phenomenon (eg, to modulate a biological activity means to increase or decrease a biological activity).

As used herein, the term "treatment" and its like terms refer to obtaining a desired pharmacological and/or physiological effect. The effect may be a prophylactic effect, ie complete or partial prevention of a disease or condition, or symptoms thereof, and/or may be a therapeutic effect, ie partial or complete cure of the condition and/or adverse effects attributable to the condition. "Treatment" as used herein encompasses any treatment of a disease or condition in a mammal, especially a human, and includes: (a) preventing a condition in an individual who may be susceptible to the condition but has not been diagnosed with the condition; (b ) inhibit the development of the condition; and/or (c) alleviate the condition, ie cause its regression.

An "effective amount" is an amount sufficient to achieve a beneficial or desired result. An effective amount can be administered in one or more administrations. An effective amount corresponds to that amount required to provide the desired average local concentration of the particular biological agent for the intended period of treatment based on the known efficacy of the particular biological agent. Dosages can be determined by those skilled in the art by conducting preliminary animal studies and generating dose-response curves, as known in the art. As is known in the art, the maximum concentration in a dose response curve will be determined by the solubility of the compound in solution and toxicity in animal models.

An effective amount additionally corresponds to that amount necessary to provide a desired average local concentration of a particular biological agent based on the expected duration of efficacy of the particular biological agent. Losses based on circulatory fluctuations caused by physical activity (eg, 10% to 90% loss) can vary depending on the individual patient and their daily activities.

The terms "individual, subject", "subject" and "patient" are used interchangeably herein to refer to mammals, including but not limited to humans, murines, apes, felines, canines, equine mammals, bovids, livestock mammals, sport mammals and pet mammals. Especially human beings.

As used herein, the terms "determine", "assess", "analyze", "measure" and "detect" refer to quantitative as well as qualitative test" and similar terms are used interchangeably. If quantitative measurement is intended, the phrase "to measure a certain amount" and similar expressions are used. If a qualitative or quantitative determination is intended, the terms "determining the extent of proliferation" or "detecting proliferation" are used.

Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this invention, chemical elements are identified according to the Periodic Table of the Elements, CAS Edition, Handbook of Chemistry and Physics, 75th Edition, inside cover, and specific functional groups are generally defined as described therein . Additionally, general principles of organic chemistry, and specific functional moieties and reactivity, are described in "Organic Chemistry," Thomas Sorrell, University Science Books, Sausalito: 1999, the entire contents of which are incorporated herein by reference.

Certain compounds of the present invention may exist in particular geometric or stereoisomeric forms. The present invention covers all such compounds falling within the scope of the present invention, including cis and trans isomers, R- and S-enantiomers, diastereomers , (D)-isomers, (L)-isomers, their racemic mixtures and other mixtures thereof. For example, other asymmetric carbon atoms may be present in substituents of alkyl groups. All such isomers and mixtures thereof are included in the present invention.

Isomeric mixtures containing any of various isomer ratios may be utilized in accordance with the present invention. For example, in the case of combining only two isomers, containing 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 96:4, 97:3, 98 Mixtures of :2, 99:1 or 100:0 isomer ratios are contemplated in the present invention. Those of ordinary skill will readily appreciate that more complex isomer mixtures encompass similar ratios.

For example, if a specific enantiomer of a compound of the invention is desired, it can be prepared by asymmetric synthesis or by derivatization with a chiral auxiliary, separation of the resulting diastereomeric mixture and cleavage of the auxiliary group to provide Pure desired enantiomer. Alternatively, if the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), diastereoisomeric salts are formed with an appropriate optically active acid or base, followed by fractional crystallization or layering as is well known in the art. The diastereoisomers thus formed are resolved analytically and the pure enantiomers are subsequently recovered.

The term "aliphatic" as used herein includes saturated and unsaturated nonaromatic straight chain (i.e. unbranched), branched chain, acyclic and cyclic (i.e. carbocyclic) hydrocarbons, optionally modified by a Or multiple functional groups are substituted. As will be appreciated by those of ordinary skill, "aliphatic" herein is intended to include, but is not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties. Thus, as used herein, the term "alkyl" includes straight chain, branched chain and cyclic alkyl groups. Similar provisions apply to other generic terms such as "alkenyl", "alkynyl" and the like. Furthermore, as used herein, the terms "alkyl," "alkenyl," "alkynyl," and similar terms encompass both substituted and unsubstituted groups. In certain embodiments, as used herein, "aliphatic" is used to indicate those aliphatic groups (cyclic, acyclic, substituted, unsubstituted, branched chain) having 1-20 carbon atoms or unbranched). Aliphatic group substituents include, but are not limited to, any of the substituents described herein that result in the formation of a stable moiety (e.g., aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclyl, aryl , heteroaryl, acyl, oxo, imino, thiooxo, cyano, isocyano, amino, azido, nitro, hydroxyl, thiol, halo, aliphatic amino, Heteroaliphatic amino, alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl, arylalkyl, aliphatic oxy, heteroaliphatic oxy, alkoxy, heteroalkane Oxy, Aryloxy, Heteroaryloxy, Aliphatic Thioxy, Heteroaliphatic Thioxy, Alkylthio, Heteroalkylthio, Arylthio, Heteroarylthio , acyloxy and the like, each of which may or may not be further substituted).

As used herein, the term "alkyl" has its ordinary meaning in the art and refers to a group of saturated aliphatic groups, including straight-chain alkyls, branched-chain alkyls, cycloalkyl (alicyclic) groups , Cycloalkyl substituted by alkyl and alkyl substituted by cycloalkyl. In some cases, the alkyl group may be a lower alkyl group, that is, an alkyl group having 1 to 10 carbon atoms (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl , nonyl or decyl). In some embodiments, a straight or branched chain alkyl group may have 30 or fewer carbon atoms in its backbone, and in some cases 20 or fewer carbon atoms. In some embodiments, a linear or branched chain alkyl group may have 12 or fewer in its backbone (e.g., C ₁ -C ₁₂ for a straight chain, C ₃ -C ₁₂ for a branched chain), 6 6 or less, or 4 or less carbon atoms. Likewise, a cycloalkyl group can have 3-10 carbon atoms in its ring structure, or 5, 6, or 7 carbons in the ring structure. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, t-butyl, cyclobutyl, hexyl, and cyclohexyl.

The term "alkylene" as used herein refers to a divalent alkyl group. "Alkylene" is polymethylene, ie -(CH ₂ ) _z -, where z is a positive integer, such as 1 to 20, 1 to 10, 1 to 6, 1 to 4, 1 to 3, 1 to 2 or 2 to 3. A substituted alkylene chain is a polymethylene group in which one or more methylene hydrogen atoms have been replaced by a substituent. Suitable substituents include those described herein for substituted aliphatic groups.

Generally, the prefix "亚" is used to describe divalent groups. Accordingly, any term defined herein may be modified with the prefix "sub" to describe the bivalent version of that moiety. For example, a divalent carbocyclic ring is "carbocyclylene", a divalent aryl ring is "arylene", a divalent benzene ring is "phenylene", and a divalent heterocyclic ring is "heterocyclylene". , a divalent heteroaryl ring is "heteroarylene", a divalent alkyl chain is "alkylene", a divalent alkenyl chain is "alkenylene", and a divalent alkynyl chain is "alkynylene" , the divalent heteroalkyl chain is "heteroalkylene", the divalent heteroalkenyl chain is "heteroalkenylene", the divalent heteroalkynyl chain is "heteroalkynylene", etc.

The terms "alkenyl" and "alkynyl" have their ordinary meanings in the art and refer to unsaturated aliphatic groups similar in length and possible substitution to the above-mentioned alkyl groups, but containing at least one double or triple bond, respectively .

In certain embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-20 aliphatic carbon atoms. In certain other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-10 aliphatic carbon atoms. In other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-8 aliphatic carbon atoms. In other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-6 aliphatic carbon atoms. In other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-4 carbon atoms. Representative aliphatic groups thus include, but are not limited to, for example methyl, ethyl, n-propyl, isopropyl, allyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n- Pentyl, sec-pentyl, isopentyl, tert-pentyl, n-hexyl, sec-hexyl, moieties and the like, which in turn may carry one or more substituents. Alkenyl groups include, but are not limited to, groups such as vinyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and the like. Representative alkynyl groups include, but are not limited to, ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like.

As used herein, the term "cycloalkyl" specifically refers to a group having 3 to 10, preferably 3 to 7 carbon atoms. Suitable cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and the like, as in the case of other aliphatic, heteroaliphatic or heterocyclic moieties Herein, it may be optionally substituted with substituents including, but not limited to: aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl; heteroarylalkyl; alkoxy; Aryloxy; Heteroalkoxy; Heteroaryloxy; Alkylthio; Arylthio; Heteroalkylthio; Heteroarylthio; -F; -Cl; -Br; -I; -OH; -NO ₂ -CN; _-CF3 ; _-CH2CF3 ; _-CHCl2 ; _-CH2OH ; _{-CH2CH2OH ; -CH2NH2 ; -CH2SO2CH3 ; -C} (O) _{R x} ; _-CO2 (Rx ₎ ;-CON(Rx) ₂ ;-OC(O ₎ Rx; _-OCO2Rx ; _-OCON (Rx _{) 2} ;-N( _Rx ) _{2 ;} -S (O) _2Rx ; _-NRx ( _CO ) _Rx , where each occurrence of Rx independently includes, but _is not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkane or heteroarylalkyl, wherein any of the aliphatic, heteroaliphatic, arylalkyl, or heteroarylalkyl substituents described above and herein may be substituted or unsubstituted , branched or unbranched, cyclic or acyclic, and wherein any of the aryl or heteroaryl substituents described above and herein may be substituted or unsubstituted. Other examples of generally applicable substituents are illustrated by the specific embodiments shown in the examples described herein.

As used herein, the term "heteroaliphatic" refers to an aliphatic moiety as defined herein, which includes saturated and unsaturated non-aromatic straight chain (i.e. unbranched), branched chain, acyclic, cyclic ( That is, heterocycles) or polycyclic hydrocarbons, which are optionally substituted with one or more functional groups, and contain one or more atoms such as oxygen, sulfur, nitrogen, phosphorus or silicon to replace carbon atoms. In certain embodiments, one or more hydrogen atoms on a heteroaliphatic moiety can be independently replaced by one or more substituents. As will be appreciated by those of ordinary skill, "heteroaliphatic" herein is intended to include, but is not limited to, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocycloalkyl, heterocycloalkenyl, and heterocycloalkynyl moieties. Thus, the term "heteroaliphatic" includes the terms "heteroalkyl," "heteroalkenyl," "heteroalkynyl," and the like. Furthermore, as used herein, the terms "heteroalkyl," "heteroalkenyl," "heteroalkynyl," and the like encompass both substituted and unsubstituted groups. In certain embodiments, as used herein, "heteroaliphatic" is used to indicate those heteroaliphatic groups (cyclic, acyclic, substituted, unsubstituted, branched or unbranched). Heteroaliphatic group substituents include, but are not limited to, any of the substituents described herein that result in the formation of a stable moiety (e.g., aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclyl, aryl radical, heteroaryl, acyl, sulfinyl, sulfonyl, oxo, imino, thioxo, cyano, isocyano, amino, azido, nitro, hydroxyl, thiol, halo , aliphatic amino, heteroaliphatic amino, alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl, arylalkyl, aliphatic oxy, heteroaliphatic oxy, alkane Oxygen, Heteroalkoxy, Aryloxy, Heteroaryloxy, Aliphatic Thioxy, Heteroaliphatic Thioxy, Alkylthio, Heteroalkylthio, Arylthio, Hetero arylsulfoxy, acyloxy and the like, each of which may or may not be further substituted).

The term "heteroalkyl" has its ordinary meaning in the art and refers to an alkyl group as described herein in which one or more carbon atoms are replaced by a heteroatom. Suitable heteroatoms include oxygen, sulfur, nitrogen, phosphorus, and the like. Examples of heteroalkyl include, but are not limited to, alkoxy, amino, thioester, poly(ethylene glycol), and alkyl-substituted amino.

The terms "heteroalkenyl" and "heteroalkynyl" have their ordinary meanings in the art and refer to unsaturated fatty acids similar in length and possible substitution to the aforementioned heteroalkyl groups, but containing at least one double or triple bond, respectively. family group.

Some examples of substituents for the aforementioned aliphatic (and other) moieties of the compounds of the present invention include, but are not limited to, aliphatic; heteroaliphatic; aryl; heteroaryl; alkylaryl; alkylheteroaryl ;Alkoxy;Aryloxy;Heteroalkoxy;Heteroaryloxy;Alkylthio;Arylthio;Heteroalkylthio;Heteroarylthio;F;Cl;Br;I;-OH;-NO _{-CH2 ; -CH2CF3 ; -CHCl2 ; -CH2OH ; -CH2CH2OH ; -CH2NH2 ; -CH2SO} -C(O)Rx; _-CO2 ( _Rx );-CON( _Rx ) _{2 ;} -OC(O) _{Rx ; -OCO2Rx ; -OCON} (Rx _{) 2} ; -N(R _x ) ₂ ; -S(O) ₂ R _x ; -NR _x (CO)R _x , where each occurrence of R _x independently includes (but is not limited to) aliphatic, alicyclic, hetero Aliphatic, heterocyclyl, aryl, heteroaryl, alkylaryl or alkylheteroaryl, wherein the aliphatic, heteroaliphatic, alkylaryl or alkyl described above and herein Any of the heteroaryl substituents may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and wherein of the aryl or heteroaryl substituents described above and herein Either may be substituted or unsubstituted. Other examples of generally applicable substituents are illustrated by the specific embodiments shown in the examples described herein.

The term "aryl" has its ordinary meaning in the art and refers to optionally substituted rings having a single ring (such as phenyl), multiple rings (such as biphenyl) or multiple fused rings, at least one of which is aromatic. Aromatic carbocyclic group of the group (such as 1,2,3,4-tetrahydronaphthyl, naphthyl, anthracenyl or phenanthrenyl). That is, at least one ring may have a conjugated pi-electron system, while the other adjacent rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, and/or heterocyclyl. As described herein, aryl groups can be optionally substituted. Substituents include, but are not limited to, any of the previously mentioned substituents, ie, for aliphatic moieties or as described for other moieties as disclosed herein, can form stable compounds. In some cases, aryl is a stable monocyclic or polycyclic unsaturated moiety, preferably having 3-14 carbon atoms, each of which may be substituted or unsubstituted. "Carbocyclic aryl" means an aryl group in which the ring atoms in the aromatic ring are carbon atoms. Carbocyclic aryl groups include monocyclic carbocyclic aryl groups and polycyclic or fused compounds (eg, two or more adjacent ring atoms are shared by two adjacent rings), such as naphthyl.

The term "heteroaryl" has its ordinary meaning in the art and refers to an aryl group comprising at least one heteroatom as a ring atom. "Heteroaryl" is a stable heterocyclic or polyheterocyclic unsaturated moiety preferably having 3-14 carbon atoms, each of which may be substituted or unsubstituted. Substituents include, but are not limited to, any of the previously mentioned substituents, ie, for aliphatic moieties or as described for other moieties as disclosed herein, can form stable compounds. In some instances, heteroaryl is a cyclic aromatic group having 5 to 10 ring atoms, one of which is selected from S, O, and N; 0, 1, or 2 ring atoms are independently selected from S , O and N other heteroatoms; and the remaining ring atoms are carbon, the group is connected to the rest of the molecule through any ring atom, such as pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl , thiazolyl, Azolyl, iso Azolyl, thiadiazolyl, Oxadiazolyl, thienyl, furyl, quinolinyl, isoquinolyl and the like.

It should also be understood that aryl and heteroaryl moieties as defined herein may be attached via an alkyl or heteroalkyl moiety and thus also include -(alkyl)aryl, -(heteroalkyl)aryl, -( Heteroalkyl)heteroaryl and -(heteroalkyl)heteroaryl moieties. Thus, as used herein, the phrase "aryl or heteroaryl moiety" is not to be confused with "aryl, heteroaryl, -(alkyl)aryl, -(heteroalkyl)aryl, -(heteroalkyl)heteroaryl radical and -(heteroalkyl)heteroaryl" are interchangeable. Substituents include, but are not limited to, any of the previously mentioned substituents, ie, for aliphatic moieties or as described for other moieties as disclosed herein, can form stable compounds.

It will be appreciated that aryl and heteroaryl groups (including bicyclic aryl groups) may be unsubstituted or substituted, wherein substitution includes replacement of one or more hydrogen atoms thereon independently with any one or more of the following moieties, including (but Not limited to): aliphatic; alicyclic; heteroaliphatic; heterocyclic; aromatic; heteroaromatic; aryl; heteroaryl; alkylaryl; heteroalkylaryl; alkyl Heteroaryl; heteroalkylheteroaryl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; F; Cl _{-CH2CF3 ; -CHCl2 ; -CH2OH ; -CH2CH2OH ; _ _ -CH2NH2 ; -CH2SO2CH3} ; _-C (O)Rx; _-CO2 ( _Rx ) _{; -CON(Rx)2 ; -OC} (O) _{Rx ; -OCO2R x} ;-OCON(R _x ) ₂ ;-N(R _x ) ₂ ;-S(O)R _x ;-S(O) ₂ R _x ;-NR _x (CO)R _x , where each occurrence of R _x independently include, but are not limited to, aliphatic, cycloaliphatic, heteroaliphatic, heterocyclyl, aromatic, heteroaromatic, aryl, heteroaryl, alkylaryl, alkylhetero Aryl, heteroalkylaryl or heteroalkylheteroaryl, wherein aliphatic, cycloaliphatic, heteroaliphatic, heterocyclyl, alkylaryl or alkylheteroaryl as described above and herein Any of the radical substituents may be substituted or unsubstituted, branched or unbranched, saturated or unsaturated, and wherein the aromatic, heteroaromatic, aryl, hetero Any of the aryl, -(alkyl)aryl, or -(alkyl)heteroaryl substituents may be substituted or unsubstituted. Additionally, it is understood that any two adjacent groups taken together may represent a 4, 5, 6 or 7 membered substituted or unsubstituted alicyclic or heterocyclic moiety. Other examples of generally applicable substituents are illustrated by the specific embodiments described herein.

The term "heterocycle" has its ordinary meaning in the art and refers to a ring containing at least one heteroatom as a ring atom, and in some cases 1 to 3 heteroatoms as ring atoms, the remaining ring atoms being carbon atoms group. Suitable heteroatoms include oxygen, sulfur, nitrogen, phosphorus, and the like. In some cases, the heterocyclic ring can be a 3 to 10 membered ring structure or a 3 to 7 membered ring structure including 1 to 4 heteroatoms.

The term "heterocycle" may include heteroaryl, saturated heterocyclyl (eg, cycloheteroalkyl), or combinations thereof. A heterocycle can be a saturated molecule or can contain one or more double bonds. In some instances, the heterocycle is a nitrogen heterocycle in which at least one ring contains at least one nitrogen ring atom. Heterocycles can be fused with other rings to form polycyclic heterocycles. Heterocyclic rings can also be fused with spirocyclic groups. In some cases, a heterocycle can be attached to the compound through a nitrogen or carbon atom in the ring.

Heterocycles include, for example, thiophene, benzothiophene, thianthrene, furan, tetrahydrofuran, pyran, isobenzofuran, chromene, dibenzopyran, thiaxanthene, pyrrole, dihydropyrrole, pyrrolidine, imidazole, Pyrazole, pyrazine, isothiazole, iso Azole, pyridine, pyrazine, pyrimidine, pyridazine, indazine, isoindole, indole, indazole, purine, quinozine, isoquinoline, quinoline, phthalazine, naphthaline, quinoxaline, quinazole phylloline, cinnoline, pteridine, carbazole, carboline, triazole, tetrazole, azole, iso Azole, thiazole, isothiazole, phenanthridine, acridine, pyrimidine, phenanthroline, phenazine, phenpyrazine, phenothiazine, furoxan, phenanthroline oxazine, pyrrolidine, oxolane, thiolane, azole, oxazine, piperidine, homopiperidine (hexamethyleneimine), piperazine (such as N-methylpiperazine), morpholine, lactone, lactam (such as azetidinone and pyrrolidone), sulphonyl Amides, sultones, their other saturated and/or unsaturated derivatives and their analogs. Heterocycles can optionally be substituted at one or more positions with substituents as described herein. In some cases, a heterocyclic ring may be bonded to the compound via a heteroatom ring atom (eg, nitrogen). In some instances, a heterocycle may be bonded to the compound via a carbon ring atom. In some cases, the heterocycle is pyridine, imidazole, pyrazine, pyrimidine, pyridazine, acridine, acridin-9-amine, bipyridine, diazine, quinoline, benzoquinoline, benzisoquinoline , phenanthridine-1,9-diamine or an analogue thereof.

As used herein, the terms "halo" and "halogen" refer to an atom selected from fluorine, chlorine, bromine and iodine.

The term "haloalkyl" denotes an alkyl group as defined above to which 1, 2 or 3 halogen atoms are attached and examples are groups such as chloromethyl, bromoethyl, trifluoromethyl and the like.

As used herein, the term "amino" refers to a primary amine (-NH ₂ ), a secondary amine (-NHR _x ), a tertiary amine (-NR _x R _y ) or a quaternary ammonium (-N ⁺ R _x R _y R _z ), wherein R _x , R _y and R _z are independently aliphatic, cycloaliphatic, heteroaliphatic, heterocyclic, aryl or heteroaryl moieties as defined herein. Examples of amino groups include, but are not limited to, methylamino, dimethylamino, ethylamino, diethylamino, methylethylamino, isopropylamino, N-piperidinyl, trimethylamino, and propylamino.

The term "alkyne" has its ordinary meaning in the art and refers to a branched or unbranched unsaturated hydrocarbon radical containing at least one triple bond. Non-limiting examples of alkynes include acetylene, propyne, 1-butyne, 2-butyne, and the like. Alkyne groups may be substituted and/or have one or more hydrogen atoms replaced by functional groups such as hydroxyl, halogen, alkoxy and/or aryl.

As used herein, the term "alkoxy" (or "alkyloxy") or "sulfanyl" refers to the parent molecular moiety attached via an oxygen atom or via a sulfur atom to an alkyl group as previously defined. In certain embodiments, the alkyl group contains 1-20 aliphatic carbon atoms. In certain other embodiments, the alkyl group contains 1-10 aliphatic carbon atoms. In other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-8 aliphatic carbon atoms. In other embodiments, the alkyl group contains 1-6 aliphatic carbon atoms. In other embodiments, the alkyl group contains 1-4 aliphatic carbon atoms. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, tert-butoxy, neopentyloxy, and n-hexyloxy. Examples of sulfanyl groups include, but are not limited to, methylthio, ethylthio, propylthio, isopropylthio, n-butylthio, and the like.

The term "aryloxy" refers to the group -O-aryl. The term "acyloxy" refers to the group -O-acyl.

The term "alkoxyalkyl" refers to an alkyl group substituted with at least one alkoxy group (eg, 1, 2, 3 or more alkoxy groups). For example, alkoxyalkyl can be optionally substituted -(C _1-6 alkyl)-O-(C _1-6 alkyl). In some cases, the alkoxyalkyl group is optionally replaced by another optionally substituted alkoxyalkyl group (eg, -(C _1-6 alkyl)-O-(C _1-6 alkyl)-O -(C _1-6 alkyl)) substitution.

It should be understood that the aforementioned groups and/or compounds as described herein may be optionally substituted with any number of substituents or functional moieties. That is, any of the above groups may be optionally substituted. As used herein, the term "substituted" is intended to include all permissible substituents of organic compounds, "permitted" under the chemical rules of valence known to those of ordinary skill. In general, the term "substituted", whether preceded by the term "optionally" or not, and the substituents contained in the formulas of the present invention, means that the hydrogen groups in the given structure are replaced by the specified substituents. When more than one position in any given structure may be substituted with one or more substituents selected from a particular group, the substituents at each position may be the same or different. It should be understood that "substituted" also includes substitutions that result in stable compounds, such as substitutions that do not spontaneously undergo transformations such as rearrangements, cyclizations, removals, and the like. In some instances, "substituted" may generally refer to the replacement of a hydrogen with a substituent as described herein. However, as used herein, "substituted" does not encompass the replacement and/or alteration of a key functional group by which a molecule is identified, eg such that a "substituted" functional group becomes a different functional group through substitution. For example, a "substituted phenyl" must still contain a phenyl moiety and cannot be modified by substitution, in this definition, to eg a pyridine ring. In a broad embodiment, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Representative substituents include, for example, those described herein. The permissible substituents may be one or more and the same or different for appropriate organic compounds. For purposes of the present invention, heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein that satisfy the valences of the heteroatoms. Furthermore, it is in no way intended that this invention be limited by the permissible substituents of organic compounds. Combinations of substituents and variables envisioned by this invention are preferably those that result in the formation of stable compounds useful in the formation of developers or developer precursors. As used herein, the term "stable" preferably means that the compound is sufficiently stable to permit manufacture and maintain the integrity of the compound for a sufficient period of time to be detected and preferably for a sufficient period of time to be suitable for the purposes detailed herein.

Examples of substituents include, but are not limited to, halogen, azido, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, alkoxy, amino, nitro, mercapto, imino, Amide, phosphonate, phosphonite, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamide, ketone, aldehyde, ester, heterocyclic, aromatic or heteroaromatic moiety, _-CF3 , -CN, aryl, aryloxy, perhaloalkoxy, aralkoxy, heteroaryl, heteroaryloxy, heteroarylalkyl, heteroaralkoxy radical, azido, amino, halo, alkylthio, oxo, acylalkyl, carboxyl ester, -carboxamido, acyloxy, aminoalkyl, alkylaminoaryl, alkylaryl, alkyl Aminoalkyl, alkoxyaryl, arylamino, aralkylamino, alkylsulfonyl, -carboxamidoalkylaryl, -carboxamidoaryl, hydroxyalkyl, haloalkyl, alkane Aminoalkylcarboxy-, aminocarboxamidoalkyl-, cyano, alkoxyalkyl, perhaloalkyl, arylalkoxyalkyl and the like.

Any of the compounds described herein may be in various forms such as, but not limited to, salts, solvates, hydrates, tautomers, and isomers.

As used herein, the term "pharmaceutically acceptable salt" means, within the scope of sound medical judgment, suitable for use in contact with tissues of humans and lower animals without undue toxicity, irritation, allergic reactions and the like, and Take those with a grain of salt commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19 (incorporated herein by reference). Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are amino acids and inorganic acids (such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid) or organic acids (such as acetic acid, oxalic acid, maleic acid) , tartaric acid, citric acid, succinic acid, or malonic acid), or salts formed by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, besylate, benzoate, bisulfate, borate, butyrate, camphoric acid Salt, Camphor Sulfonate, Citrate, Cyclopentane Propionate, Digluconate, Lauryl Sulfate, Ethane Sulfonate, Formate, Fumarate, Glucose Enanthate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, lauric acid Salt, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate Salt, Palmitate, Pamoate, Pectate, Persulfate, 3-Phenylpropionate, Phosphate, Picrate, Pivalate, Propionate, Stearate, Succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate and similar salts. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N ⁺ (C _1-4 alkyl) ₄ salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Other pharmaceutically acceptable salts include, where appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, Low carbon alkyl sulfonate and aryl sulfonate.

In certain embodiments, the compounds are in the form of hydrates or solvates. As used herein, the term "hydrate" refers to a compound that is non-covalently associated with one or more water molecules. Likewise, the term "solvate" refers to a compound that is non-covalently associated with one or more molecules of an organic solvent.

In certain embodiments, the compounds described herein can exist in various tautomeric forms. As used herein, the term "tautomer" includes two or more species due to at least one shift in hydrogen atom form and at least one change in valency (e.g., single bond to double bond, double bond to single bond, or vice versa). Compounds that can be transformed into each other. The exact ratio of tautomers depends on several factors including temperature, solvent and pH. Tautomerization (ie, a reaction affording a tautomeric pair) can be catalyzed by either an acid or a base. Exemplary tautomerizations include ketone to enol; amide to imide; lactam to lactim; enamine to imine; and enamine to (different) enamine tautomerization.

In certain embodiments, the compounds described herein can exist in various isomeric forms. As used herein, the term "isomer" includes any and all geometric isomers and stereoisomers (eg, enantiomers, diastereomers, etc.). For example, "isomer" includes within the scope of the present invention cis-isomers and trans-isomers, E-isomers and Z-isomers, R-enantiomers and S-isomers. Enantiomers, diastereomers, (D)-isomers, (L)-isomers, racemic mixtures thereof, and other mixtures thereof. For example, isomers/enantiomers may in some embodiments be provided substantially free of the corresponding enantiomer, and may also be referred to as "optically-enriched." As used herein, "optically enriched" means that a compound is composed of a significantly greater proportion of one enantiomer. In certain embodiments, compounds of the present invention consist of at least about 90% by weight of the preferred enantiomer. In other embodiments, the compounds consist of at least about 95%, 98%, or 99% by weight of the preferred enantiomer. Preferred enantiomers may be isolated from racemic mixtures by any method known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC), and formation and crystallization of chiral salts, or by different prepared by symmetrical synthesis. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen, S.H. et al., Tetrahedron 33:2725 (1977); Eliel, E.L. Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962) ; Wilen, S.H. Tables of Resolving Agents and Optical Resolutions, p. 268 (ed. E.L. Eliel, Univ. of Notre Dame Press, Notre Dame, IN 1972).

The present invention provides endogenous embryonic/fetal hemoglobin chain inducers in various formulations for therapeutic administration. In one embodiment, the agent is formulated into a pharmaceutical composition by combining with an appropriate pharmaceutically acceptable carrier or diluent, and formulated into solid, semi-solid, liquid or gaseous preparations, such as tablets, capsules, Powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosol forms. Thus, administration is accomplished in a variety of ways. In some formulations, the inducer is administered systemically; in others, the inhibitor is localized by means of the formulation, for example using an implant to retain the active dose at the implantation site.

Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

When a range of values is provided, it is understood that unless the context clearly dictates otherwise, each intervening value between the upper and lower limits of that range (to the tenth of the unit of the lower limit) and any other specified value within that stated range or Intermediate values are all encompassed within the present invention. The upper and lower limits of such smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned below are hereby incorporated by reference. Unless otherwise mentioned, the techniques employed herein are standard methods well known to those of ordinary skill in the art.

It must be noted that as used herein and in the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a biomarker" includes a plurality of such biomarkers and reference to "a sample" includes reference to one or more samples and equivalents thereof known to those skilled in the art, and the like. It should also be noted that claims may be drafted to exclude any optional elements. Accordingly, this statement is intended to serve as a precondition for the use of exclusive terms such as "solely," "only," and the like in conjunction with the recitation of claim elements of the scope of the application or the use of a "negative" limitation. Furthermore, any positively recited element of the invention provides the basis for a negative limitation excluding that element from the claimed scope.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are herein incorporated by reference Methods and/or materials in connection with which the publications are cited are disclosed and described herein. The citation of any publication is for its disclosure prior to the filing date of this application and is not to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. In addition, the dates of publication provided may differ from actual publication dates which may need to be independently confirmed.

Other objects, features and advantages of the present invention will become apparent from the following [Detailed Description of the Invention]. However, it should be understood that although the [Detailed Description of the Invention] and the specific examples indicate preferred embodiments of the present invention, they are provided by way of illustration only so that those skilled in the art will become apparent from this [Detailed Description of the Invention] the spirit and scope of the present invention Various changes and amendments.

Description of drawings

Figure 1 shows the physical map of the human α-like and β-like hemoglobin gene clusters. The human α-like and β-like hemoglobin gene clusters located on chromosomes 16 and 11, respectively, both extend over 50 kb of the genome. Each gene cluster consists of an upstream locus control region (LCR) and a downstream paralogic hemoglobin gene. The expression of individual hemoglobin genes in cells of the erythroid lineage is temporarily controlled during a developmental period called the hemoblobin switch. Hemoglobin turnover is accompanied by reciprocal silencing of embryonic/fetal hemoglobin genes and equivalent activation of fetal/adult hemoglobin genes at each developmental stage, which is also accompanied by a shift in the site of erythropoiesis.

Figure 2 shows the results of high-throughput screening of compounds capable of inducing embryonic/fetal hemoglobin genes. (A) Physical map of the dual fluorescent reporter plastid. (B) Experimental procedure for high-throughput screening. (Step 1) 10,000 heterocyclic compounds were added to a 96-well culture plate and tested for their ability to increase embryonic/fetal hemoglobin promoter at a working concentration of 10 μM. (Step 2 and Step 3) The high expression of DsRed fluorescence was detected by a fluorescence reader, and further confirmed by a digital image detector. (Step 4) Then, the compound-induced activation of endogenous embryonic/fetal hemoglobin genes was verified by real-time RT-PCR analysis. (C) Filtered data shown in bitmap. About 8 compounds out of 10,000 were found to increase DsRed fluorescence intensity by a factor of 1.5 or more. Real-time RT-PCR analysis showed that only one of the eight compounds (compound A; arrow) could induce MEL cells to express more than 10 times the expression of endogenous mouse embryonic εy hemoglobin mRNA.

Figure 3 illustrates the induction of endogenous hemoglobin genes by Compound A in primary cultures of human erythroid progenitor cells. (A) Dosage effect of Compound A in differentiating human erythroid progenitor cells. The differentiated primary culture cells were treated with different concentrations of Compound A for 3 days. Total mRNA was extracted and the relative induction of beta and gamma hemoglobin mRNA was determined by real-time RT-PCR analysis. The cell proliferation ability was measured using alamar blue reagent (as a 100% blank control). White bars indicate the relative expression of β-hemoglobin. Gray bars indicate the relative content of γ-hemoglobin mRNA. (B) Comparison of gamma and beta hemoglobin gene expression patterns in cultured primary erythroid cells treated with different fetal hemoglobin inducers. The cells were treated with compound A (0.25 μM), TSA (0.33 μM), hydroxyurea (HU; 100 μM) and sodium butyrate (NaB; 0.5 mM) for 3 days, respectively. Total RNA was then isolated for real-time RT-PCR analysis. The relative induction of the beta hemoglobin gene (white bars) and the gamma hemoglobin gene (gray bars) was then determined.

Figure 4 shows the relative expression levels of transcription factors related to the regulation of hemoglobin genes. Primary human erythroid cultures were treated with 0.25 μM Compound A, and then the relative expression levels of different transcription factors were determined by real-time RT-PCR analysis. The white bars indicate the performance of the blank control set to 1. Black bars are relative expression in Compound A-treated cells.

Figure 5 illustrates the induction of fetal gamma globulin gene expression by six heterocyclic compounds having a common core structure. (A) Several heterocyclic compounds with a common naphthoylbenzimidazole core structure were selected to test their ability to induce the expression of the gamma hemoglobin gene in human primary cultured erythroid cells. The induction of the gamma-globulin gene by these compounds was then analyzed by real-time RT-PCR (black bars). Cell proliferation ability (curve) was measured using Alamar blue reagent (as a 100% blank control). (B) Shows the common core structure (benzo[de]-benzo[4,5]imidazol[2,1-a]isoquinolin-7-one) and its position numbers for possible modification with branched side chains.

Figure 6 illustrates the treatment of primary erythroid cultures with different fetal gamma-globulin inducers (hydroxyurea (HU), sodium butyrate (NaB) and the provided naphthoylbenzimidazole compound) at _IC503 Days later, in which induction of endogenous hemoglobin genes occurs. Total mRNA was extracted and the relative induction of beta and gamma hemoglobin mRNA was determined by real-time RT-PCR analysis. White bars indicate the relative expression of β-hemoglobin. Gray bars indicate the relative content of γ-hemoglobin mRNA.

Figure 7 shows the induction of fetal gamma hemoglobin gene expression in primary cultures of human erythroid cells by the specific CaMKK inhibitor STO-609. The dose effects of STO-609 on the proliferation of differentiated human erythroid progenitor cells and gamma/beta hemoglobin gene expression were analyzed according to the methods described for compounds A-F. The differentiated primary cultured erythroid cells were treated with different concentrations of STO-609 for 3 days. (A) Relative induction of β (white bars) and γ hemoglobin mRNA (black bars) was determined by real-time RT-PCR analysis. The cell proliferation curve is shown with the blank control as 100%. (B) The relative expression of NF-E4 was determined by real-time RT-PCR analysis and is shown by gray bars. (C) shows the relative expression of Bcl11a in response to treatment with different concentrations of STO-609 (white bars). (D) shows the relative expression of c-Myb in cells treated with different concentrations of STO-609 (black bars). The blank control was set to 1.

Figure 8 shows a draft model of a possible mechanism of how heterocyclic compounds with the same core structure activate fetal gamma globulin gene expression. Our studies have shown that the induction of fetal γ-globulin gene by these heterocyclic compounds is mediated at least in part by regulating the expression of several transcription factors, including up-regulation of NF-E4 expression and down-regulation of Bcl11a and c-Myb. These heterocyclic compounds (such as STO-609) may also interfere with the activation of MEK/Erk signaling via the CaMKK/CaMK1 pathway. CaMKK-inhibitory features of heterocyclic compounds with core structure (benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one) may result in inhibition of gamma hemoglobin gene transcription the induction.

Figure 9 shows the absolute quantification of [beta]-like hemoglobin mRNA content in primary erythroid cultures. With compound A (0.2 μM), compound B (21.8 μM), compound C (75.2 μM), compound F (103 μM), hydroxyurea (HU; 146.5 μM) or sodium butyrate (NaB; 221.5 μM) under IC ₅₀ Differentiated human primary erythroid cells were treated for 3 days, and mRNA levels were analyzed by real-time RT-PCR analysis. The number of copies of each hemoglobin mRNA per microgram of total mRNA is estimated and displayed. Brackets indicate the relative proportion (%) of individual hemoglobin chains in total β-like hemoglobin mRNA.

Fig. 10 shows the comparison of IC ₅₀ and γ-globulin gene induction ability of 6 heterocyclic compounds, sodium butyrate and hydroxyurea. The induction factor of γ-globulin gene expression was determined by real-time RT-PCR analysis of mRNA of human primary erythroid cultures treated or not treated with six heterocyclic compounds, butyric acid and hydroxyurea. _IC50 values are the concentrations of individual compounds that reduce the rate of cell proliferation by 50%. The effective concentration (EC) was defined as the concentration of the compound that induced expression of the gamma globulin gene by 1.88-fold (ie, the multiple of the gamma globulin gene induced by hydroxyurea at its _IC50 ). The therapeutic window was calculated as the ratio of _IC50 to EC ( _IC50 /EC), which reflects the benefit of heterocyclic compounds relative to hydroxyurea.

Detailed description of the invention

Part of the present invention is the identification of small molecules that induce gamma globulin gene expression. The compounds identified herein are useful in the treatment of beta-thalassemia or sickle cell anemia via induction of endogenous embryonic/fetal hemoglobin chains.

Compounds of interest are naphthoylbenzimidazoles, eg, compounds comprising a naphthoylbenzimidazole backbone substituted with one or more substituents. Any of positions 1, 2, 3, 4, 5, 6, 7, 8, 9 and/or 10 of the naphthoylbenzimidazole skeleton of formula (I) may be substituted:

In certain embodiments, positions 3 and 4 of the naphthoylbenzimidazole backbone are substituted.

In certain embodiments, substituents may cause optical isomerism and/or stereoisomerism of the compound. Salt, solvate, hydrate and prodrug forms of the compounds are also contemplated. The present invention encompasses all such forms. Accordingly, the compounds described herein include salts, solvates, hydrates, prodrugs and isomeric forms thereof, including pharmaceutically acceptable salts, solvates, hydrates, prodrugs and isomers thereof. In certain embodiments, the compounds are metabolized to pharmaceutically active derivatives.

In certain embodiments, the present invention employs compounds of formula (I-a):

or a pharmaceutically acceptable salt, solvate or hydrate thereof,

in:

R ¹ , R ² , R ³ , R ⁴ , R ^6a and R ^6b are each independently selected from: hydrogen, halogen, alkyl, alkenyl, alkynyl, heterocyclyl, aryl, -OR ^A , -OC( O)R ^A , -SR ^A , -N(R ^B ) ₂ , -N(R ^A )C(O)R ^A , -C(O)N(R ^B ) ₂ , -CN, -NO ₂ , - ^C (O) ^RA , -C(O)ORA, -S(O) ^RA , ^-SO2RA , _{-SO2N (} ^RB ) ₂ and ^-NHSO2RB _;

Each R ⁵ is independently selected from: hydrogen, halogen, alkyl, alkenyl, alkynyl, heterocyclyl, aryl, -OR ^A , -OC(O) ^{RA , -SR A ,} -N(R ^B ) ₂ , -N(R ^A )C(O)R ^A , -C(O)N(R ^B ) ₂ , -CN, -NO ₂ , -C(O)R ^A , -C(O)OR ^A , -S(O) ^RA , ^-SO2RA , _-SO2N ( ^{RB )} ₂ and _{-NHSO2RB ;}

Each ^RA is independently selected from: hydrogen, alkyl, alkenyl, alkynyl, heterocyclyl, and aryl;

Each R ^B is independently selected from: hydrogen, alkyl, alkenyl, alkynyl, heterocyclyl and aryl, or two R ^B together with an intervening nitrogen form a heterocycle;

n is 0, 1, 2, 3 or 4.

In some embodiments, R ^6a and R ^6b are each hydrogen. In certain embodiments, the present invention employs compounds of formula (II):

or a pharmaceutically acceptable salt, solvate or hydrate thereof, wherein R ¹ , R ² , R ³ , R ⁴ and R ⁵ are as described herein.

As generally stated above, R ¹ is hydrogen, halogen, alkyl, alkenyl, alkynyl, heterocyclyl, aryl, -OR ^A , -OC(O) ^{RA , -SR A ,} -N(R ^B ) ₂ , -N(R ^A )C(O)R ^A , -C(O)N(R ^B ) ₂ , -CN, -NO ₂ , -C(O)R ^A , -C(O)OR ^A , ^-S (O) ^RA , ^-SO2RA , _-SO2N ( ^RB _{)2 ,} or _-NHSO2RB . In certain embodiments, R is selected from -H, -OH, -Cl, -Br, -F, C ^1-6 _{alkyl, C 1-6 alkoxy} , alkynyl, aryl, _-NO , -N(R ^B ) ₂ , -C(O)CH ₃ , -CO ₂ H, -C(O)OR ^A , -C(O)N(R ^B ) ₂ , -CN, heterocyclyl, - SO ₂ -alkyl and -SO ₂ -aryl. ^In certain embodiments, R is selected from -H, -OH, -Cl, -Br, -F, methyl, ethyl, methoxy, ethoxy, -C≡C-aryl, phenyl , naphthyl, -NO ₂ , -NH-C _1-6 alkyl, -C(O)CH ₃ , -CO ₂ H, -CO ₂ Et, -CONH-aryl, -CN, N-morpholinyl , -SO ₂ -alkyl and -SO ₂ -aryl. In certain embodiments, R ¹ is hydrogen. In some embodiments, R ¹ is C _1-6 alkyl. In certain embodiments, R ¹ is -C(O)OR ^A . ^In certain embodiments, R1 is _-CO2H . In certain embodiments, R ¹ is -C(O)N( ^RB ) ₂ or -N( ^RA )C(O) ^RA . In some embodiments, R ¹ is halogen. In certain embodiments, R ¹ is fluoro. In certain embodiments, R ¹ is chloro. In some embodiments, R ¹ is -N( ^RB ) ₂ . In some embodiments, R ¹ is -NH-C _1-6 alkyl. In certain embodiments, R ¹ is -NHCH ₃ . In certain embodiments, R ¹ is -NO ₂ . In some embodiments, R ¹ is optionally substituted naphthyl.

As generally stated above, R is ^hydrogen , halogen, alkyl, alkenyl, alkynyl, heterocyclyl, aryl, -OR ^A , -OC(O) ^{RA , -SR A ,} -N(R ^B ) ₂ , -N(R ^A )C(O)R ^A , -C(O)N(R ^B ) ₂ , -CN, -NO ₂ , -C(O)R ^A , -C(O)OR ^A , ^-S (O) ^RA , ^-SO2RA , _-SO2N ( ^RB _{)2 ,} or _-NHSO2RB . In certain embodiments, R is selected from -H, ^-OH , -Cl, -Br, -F, C _1-6 alkyl, C _1-6 alkoxy, alkynyl, aryl, -NO ₂ , -N(R ^B ) ₂ , -C(O)CH ₃ , -CO ₂ H, -C(O)OR ^A , -C(O)N(R ^B ) ₂ , -CN, heterocyclyl, - SO ₂ -alkyl and -SO ₂ -aryl. In certain embodiments, R is selected from -H, ^-OH , -Cl, -Br, -F, methyl, ethyl, methoxy, ethoxy, -C≡C-aryl, phenyl , naphthyl, -NO ₂ , -NH-C _1-6 alkyl, -C(O)CH ₃ , -CO ₂ H, -CO ₂ Et, -CONH-aryl, -CN, N-morpholinyl , -SO ₂ -alkyl and -SO ₂ -aryl. In certain embodiments, R is ^hydrogen . In some embodiments, R ² is C _1-6 alkyl. In certain embodiments, R ² is -C(O)OR ^A . _In certain embodiments, R2 is ^-CO2H . In certain embodiments, R ² is -C(O)N( ^RB ) ₂ or -N( ^RA )C(O) ^RA . In some embodiments, R ² is halogen. ^In certain embodiments, R is fluoro. In certain embodiments, R ² is chloro. In some embodiments, R ² is -N( ^RB ) ₂ . In some embodiments, R ² is -NH-C _1-6 alkyl. In certain embodiments, R ² is -NHCH ₃ . In certain embodiments, R ² is -NO ₂ . ^In some embodiments, R is optionally substituted naphthyl.

As generally stated above, ^R3 is hydrogen, halogen, alkyl, alkenyl, alkynyl, heterocyclyl, aryl, -OR ^A , -OC(O) ^{RA , -SR A ,} -N(R ^B ) ₂ , -N(R ^A )C(O)R ^A , -C(O)N(R ^B ) ₂ , -CN, -NO ₂ , -C(O)R ^A , -C(O)OR ^A , ^-S (O) ^RA , ^-SO2RA , _-SO2N ( ^RB _{)2 ,} or _-NHSO2RB . In certain embodiments, R ³ is selected from -H, -OH, -Cl, -Br, -F, C _1-6 alkyl, C _1-6 alkoxy, alkynyl, aryl, -NO ₂ , -N(R ^B ) ₂ , -C(O)CH ₃ , -CO ₂ H, -C(O)OR ^A , -C(O)N(R ^B ) ₂ , -CN, heterocyclyl, - SO ₂ -alkyl and -SO ₂ -aryl. In certain embodiments, ^R is selected from -H, -OH, -Cl, -Br, -F, methyl, ethyl, methoxy, ethoxy, -C≡C-aryl, phenyl , naphthyl, -NO ₂ , -NH-C _1-6 alkyl, -C(O)CH ₃ , -CO ₂ H, -CO ₂ Et, -CONH-aryl, -CN, N-morpholinyl , -SO ₂ -alkyl and -SO ₂ -aryl. In certain embodiments, R ³ is hydrogen. In some embodiments, R ³ is C _1-6 alkyl. In certain embodiments, R ³ is -C(O)OR ^A . In certain embodiments, ^R3 is _-CO2H . In certain embodiments, R ³ is -C(O)N( ^RB ) ₂ or -N( ^RA )C(O) ^RA . In some embodiments, R ³ is halogen. In certain embodiments, R ³ is fluoro. In certain embodiments, R ³ is chloro. In some embodiments, R ³ is -N( ^RB ) ₂ . In some embodiments, R ³ is -NH-C _1-6 alkyl. In certain embodiments, R ³ is -NHCH ₃ . In certain embodiments, R ³ is -NO ₂ . In some embodiments, ^R3 is optionally substituted naphthyl.

As generally stated above, R4 is hydrogen, halogen, alkyl, alkenyl, alkynyl, heterocyclyl, aryl, -OR ^A , -OC(O) ^RA , -SR ^A , -N( ^{R B} ) ₂ , -N(R ^A )C(O)R ^A , -C(O)N(R ^B ) ₂ , -CN, -NO ₂ , -C(O)R ^A , -C(O)OR ^A , ^-S (O) ^RA , ^-SO2RA , _-SO2N ( ^RB _{)2 ,} or _-NHSO2RB . In certain embodiments, R ⁴ is selected from -H, -OH, -Cl, -Br, -F, C _1-6 alkyl, C _1-6 alkoxy, alkynyl, aryl, -NO ₂ , -N(R ^B ) ₂ , -C(O)CH ₃ , -CO ₂ H, -C(O)OR ^A , -C(O)N(R ^B ) ₂ , -CN, heterocyclyl, - SO ₂ -alkyl and -SO ₂ -aryl. ^In certain embodiments, R is selected from -H, -OH, -Cl, -Br, -F, methyl, ethyl, methoxy, ethoxy, -C≡C-aryl, phenyl , naphthyl, -NO ₂ , -NH-C _1-6 alkyl, -C(O)CH ₃ , -CO ₂ H, -CO ₂ Et, -CONH-aryl, -CN, N-morpholinyl , -SO ₂ -alkyl and -SO ₂ -aryl. ^In certain embodiments, R4 is hydrogen. In some embodiments, R ⁴ is C _1-6 alkyl. In certain embodiments, R ⁴ is -C(O)OR ^A . ^In certain embodiments, R4 is _-CO2H . In certain embodiments, R ⁴ is -C(O)N( ^RB ) ₂ or -N( ^RA )C(O) ^RA . In some embodiments, R ⁴ is halogen. In certain embodiments, R ⁴ is fluoro. In certain embodiments, R ⁴ is chloro. In some embodiments, R ⁴ is -N( ^RB ) ₂ . In some embodiments, R ⁴ is -NH-C _1-6 alkyl. In certain embodiments, R ⁴ is -NHCH ₃ . In certain embodiments, R ⁴ is -NO ₂ . ^In some embodiments, R4 is optionally substituted naphthyl.

As generally stated above, R ⁵ is hydrogen, halogen, alkyl, alkenyl, alkynyl, heterocyclyl, aryl, -OR ^A , -OC(O) ^{RA , -SR A ,} -N(R ^B ) ₂ , -N(R ^A )C(O)R ^A , -C(O)N(R ^B ) ₂ , -CN, -NO ₂ , -C(O)R ^A -C(O)OR ^A , ^-S (O) ^RA , ^-SO2RA , _-SO2N ( ^RB _{)2 ,} or _-NHSO2RB . ^In certain embodiments, R is selected from -H, -OH, -Cl, -Br, -F, C _1-6 alkyl, C _1-6 alkoxy, alkynyl, aryl, -NO ₂ , -N(R ^B ) ₂ , -C(O)CH ₃ , -CO ₂ H, -C(O)OR ^A , -C(O)N(R ^B ) ₂ , -CN, heterocyclyl, - SO ₂ -alkyl and -SO ₂ -aryl. ^In certain embodiments, R is selected from -H, -OH, -Cl, -Br, -F, methyl, ethyl, methoxy, ethoxy, -C≡C-aryl, phenyl , naphthyl, -NO ₂ , -NH-C _1-6 alkyl, -C(O)CH ₃ , -CO ₂ H, -CO ₂ Et, -CONH-aryl, -CN, N-morpholinyl , -SO ₂ -alkyl and -SO ₂ -aryl. ^In certain embodiments, R is hydrogen. In some embodiments, R ⁵ is C _1-6 alkyl. In certain embodiments, R ⁵ is -C(O)OR ^A . ^In certain embodiments, R5 is _-CO2H . In certain embodiments, R ⁵ is -C(O)N( ^RB ) ₂ or -N( ^RA )C(O) ^RA . In some embodiments, R ⁵ is halogen. In certain embodiments, R ⁵ is fluoro. In certain embodiments, R ⁵ is chloro. In some embodiments, R ⁵ is -N( ^RB ) ₂ . In some embodiments, R ⁵ is -NH-C _1-6 alkyl. In certain embodiments, R ⁵ is -NHCH ₃ . ^In certain embodiments, R5 is _-NO2 . ^In some embodiments, R is optionally substituted naphthyl.

In some embodiments, at least two of R ¹ , R ² , R ³ and R ⁴ are hydrogen. In certain embodiments, R ¹ and R ⁴ are hydrogen. ^In certain embodiments, ^R3 and R4 are hydrogen. ^In certain embodiments, R1 and R2 are ^hydrogen . In some embodiments, at least two of R ¹ , R ² , R ³ , and R ⁴ are each hydrogen and R ⁵ is hydrogen.

In some embodiments, the compound has formula (III), (IV) or (V):

or a pharmaceutically acceptable salt, solvate or hydrate thereof, wherein R ¹ , R ² , R ³ , R ⁴ and R ⁵ are as defined herein.

In some embodiments, R is hydrogen, resulting in ^formula (III-a), (IV-a) or (Va):

or a pharmaceutically acceptable salt, solvate or hydrate thereof, wherein R ¹ , R ² , R ³ and R ⁴ are as defined herein.

In certain embodiments, one of R or ^R is ^hydrogen , resulting in formula (III-b) or (III-c):

In some embodiments, one of R ¹ , R ² , R ³ and R ⁴ has the following structure:

wherein R ¹² and R ¹³ are independently selected from hydrogen, -CO ₂ H, -C(O)OR ^A , -C(O)N(R ^B ) ₂ , -OH, -NO ₂ , -CN, halogen, Alkyl, aryl and heterocyclyl, or R ¹² and R ¹³ together with intervening atoms form a carbocyclic or heterocyclic ring; and R ¹⁴ is selected from hydrogen, -CO ₂ H, -C(O)OR ^A , -C(O)N(R ^B ) ₂ , -OH, -NO ₂ , -CN, halogen, alkyl, aryl and heterocyclyl. In certain embodiments, R ¹² and R ¹³ together with intervening atoms form a carbocyclic or heterocyclic ring. In certain embodiments, R ¹² and R ¹³ are taken together with intervening atoms to form a heterocyclic ring (eg, succinimide).

In certain embodiments, ^R12 is _-CO2H . In certain embodiments, ^R13 is _-CO2H . In certain embodiments, ^R14 is _-CO2H .

In certain embodiments, each of R ¹² and R ¹³ is -CO ₂ H.

In certain embodiments, each of R ¹² , R ¹³ and R ¹⁴ is -CO ₂ H.

In certain embodiments, one of R ¹ , R ² , R ³ and R ⁴ has the following structure:

Wherein R ¹⁴ is hydrogen, -CO ₂ H, -C(O)OR ^A , -C(O)N(R ^B ) ₂ , -OH, -NO ₂ , -CN, halogen, alkyl, aryl and hetero and R ^is selected from hydrogen, acyl, alkyl, aryl and heterocyclyl. In certain embodiments, ^R14 is _-CO2H . In certain embodiments, ^R14 is hydrogen. In certain embodiments, ^R15 is hydrogen. In certain embodiments, R ¹⁵ is alkyl. In certain embodiments, R ¹⁵ is acyl.

In certain embodiments, the compound has the structure of Formula (VI) or (VII):

or a pharmaceutically acceptable salt, solvate or hydrate thereof,

wherein R ¹² , R ¹³ and R ¹⁴ are each independently selected from hydrogen, halogen, alkyl, alkenyl, alkynyl, heterocyclyl, aryl, -OR ^A , -OC(O) ^{RA , -SR A ,} -N(R ^B ) ₂ , -N(R ^A )C(O)R ^A , -C(O)N(R ^B ) ₂ , -CN, -NO ₂ , -C(O)R ^A , -C (O)OR ^A , -S(O) ^RA , -SO ₂ ^RA , -SO ₂ N(RB ) ₂ and -NHSO ₂ ^{R B ;} or R ¹² and R ¹³ together with intervening atoms Carbocycle or heterocycle;

each ^RA is independently selected from hydrogen, alkyl, alkenyl, alkynyl, heterocyclyl, and aryl; and

Each R ^B is independently selected from hydrogen, alkyl, alkenyl, alkynyl, heterocyclyl, and aryl, or two R ^B together with an intervening nitrogen form a heterocycle.

In certain embodiments, for formula (VI) or (VII), R ¹ , R ³ , R ⁵ , R ¹² , R ¹³ and R ¹⁴ are each independently selected from -H, -OH, -Cl, -Br , -F, C _1-6 alkyl, C _1-6 alkoxy, alkynyl, aryl, -NO ₂ , -N(R ^B ) ₂ , -C(O)CH ₃ , -CO ₂ H, -C(O)OR ^A , -C(O)N(R ^B ) ₂ , -CN, heterocyclyl, -SO ₂ -alkyl and -SO ₂ -aryl. In certain embodiments, R ¹ , R ³ , R ⁵ , R ¹² , R ¹³ and R ¹⁴ are each independently selected from -H, -OH, -Cl, -Br, -F, methyl, ethyl, Methoxy, Ethoxy, -C≡C-Aryl, Phenyl, Naphthyl, -NO ₂ , -NH-C _1-6 Alkyl, -C(O)CH ₃ , -CO ₂ H, - _CO2Et , -CONH-aryl, -CN, N-morpholinyl, _-SO2 -alkyl and _-SO2 -aryl.

In certain embodiments, R ¹ , R ³ , R ⁵ , R ¹² , R ¹³ , and R ¹⁴ are each independently selected from hydrogen, —CO ₂ H, —C(O)OR ^A , —C(O)N (R ^B ) ₂ , -OH, -NO ₂ -CN, halogen, alkyl, aryl and heterocyclyl. In certain embodiments, R ¹² and R ¹³ are taken together with intervening atoms to form an optionally substituted heterocyclic ring. In certain embodiments, R ¹² and R ¹³ are taken together with intervening atoms to form a succinimidyl ring.

In certain embodiments, each of R ¹² and R ¹³ is -CO ₂ H. In certain embodiments, each of R ¹² , R ¹³ and R ¹⁴ is -CO ₂ H. In certain embodiments, each of R ³ , R ¹² , R ¹³ and R ¹⁴ is —CO ₂ H. In certain embodiments, each of R ¹ , R ¹² and R ¹³ is —CO ₂ H. In certain embodiments, each of R ¹ , R ¹² , R ¹³ and R ¹⁴ is —CO ₂ H. In certain embodiments, ^R15 is hydrogen.

In certain embodiments, R ¹² and R ¹³ are each -CO ₂ H, and R ¹⁵ is hydrogen. In certain embodiments, each of R ¹² , R ¹³ and R ¹⁴ is —CO ₂ H, and R ¹⁵ is hydrogen. In certain embodiments, R ³ , R ¹² , R ¹³ , and R ¹⁴ are each —CO ₂ H, and R ¹⁵ is hydrogen. In certain embodiments, each of R ¹ , R ¹² and R ¹³ is —CO ₂ H, and R ¹⁵ is hydrogen. In certain embodiments, each of R ¹ , R ¹² , R ¹³ and R ¹⁴ is —CO ₂ H, and R ¹⁵ is hydrogen.

In certain embodiments, the compound used in the present invention is a compound of formula (Ia), (II), (III), (IV), (V), (VI) or (VII), wherein R ² is not -C (O)R ^A .

In certain embodiments, the compound used in the present invention is a compound of formula (Ia), (II), (III), (IV), (V), (VI) or (VII), wherein R ² is not -C (O) _CH3 .

In certain embodiments, the compound used in the present invention is a compound of formula (Ia), (II), (III), (IV), (V), (VI) or (VII), wherein R ³ is not -NH - Allyl.

In certain embodiments, the compound used in the present invention is a compound of formula (Ia), ( ^II ), (III), (IV), (V), (VI) or (VII), wherein R is not -NHCH _{2CH2OH .}

In certain embodiments, the compound used in the present invention is a compound of formula (Ia), (II), (III), ( ^IV ), (V), (VI) or (VII ⁾ , wherein R and R are not is -NO ₂ .

In certain embodiments, the compound used in the present invention is a compound of formula (Ia), (II), (III), (IV), (V), (VI) or (VII), wherein when n is 2, Each R ⁵ is not simultaneously methyl.

In certain embodiments, the compound of the invention is one of the following:

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

pharmaceutical composition

The pharmaceutical compositions of the invention and the pharmaceutical compositions used according to the invention may comprise pharmaceutically acceptable excipients or carriers. As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" means any type of non-toxic inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation Auxiliary. Remington's Pharmaceutical Sciences, 16th Edition, E.W. Martin (Mack Publishing Co., Easton, PA, 1980) discloses various excipients for formulating pharmaceutical compositions and known techniques for their preparation. Some examples of materials that can be used as pharmaceutically acceptable carriers are sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose, cellulose and acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and Soybean oil; glycols, such as propylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; detergents, such as Tween80; buffers, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water ; isotonic saline; Ringer's solution; ethanol; and phosphate buffered saline, and other nontoxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, mold release agents , coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions according to the judgment of the formulator. The pharmaceutical composition of the present invention can be administered to humans and/or animals orally, rectally, parenterally, intracisternally, intravaginally, intranasally, intraperitoneally, topically (by powder, cream, ointment or drops), bucally medicine, or as a mouth spray or nasal spray.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient (i.e., microparticles, nanoparticles, liposomes, micelles, polynucleotide/lipid complexes), liquid dosage forms may contain inert diluents commonly used in the art, such as water or other Solvents, solubilizers and emulsifiers such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, dimethylformamide, oil ( Specifically, fatty acid esters of cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol, and sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Injectable preparations, such as sterile injectable aqueous or oleaginous suspensions, can be formulated according to known techniques using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are well known for use as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. In certain embodiments, the particles are suspended in a carrier fluid comprising 1 w/v% sodium carboxymethylcellulose and 0.1% by volume Tween 80.

The injectable preparations can be sterilized, for example, by filtration through a bacteria-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile water prior to use. In a sterile injectable medium.

In order to prolong the action of a drug, it is often necessary to slow the absorption of the drug from subcutaneous or intramuscular injection. This can be achieved by using liquid suspensions or crystalline or amorphous materials with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is brought about by dissolving or suspending the drug in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.

Compositions for rectal or vaginal administration are preferably suppositories, which can be prepared by mixing the granules with a suitable non-irritating excipient or carrier such as cocoa butter, polyethylene glycol or a suppository wax. , such suitable non-irritating excipients or carriers are solid at ambient temperature but liquid at body temperature and thus melt and release the particles in the rectum or vaginal cavity.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the particles are mixed with at least one pharmaceutically acceptable inert excipient or carrier such as sodium citrate or dicalcium phosphate and/or: a) a filler or bulking agent, Such as starch, lactose, sucrose, glucose, mannitol and silicic acid; b) binders such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and acacia; c) humectants , such as glycerin; d) disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; e) dissolution delaying agents, such as paraffin; f) absorption enhancers, such as four g) humectants such as cetyl alcohol and glyceryl monostearate; h) adsorbents such as kaolin and bentonite; and i) lubricants such as talc, calcium stearate, magnesium stearate, Solid polyethylene glycol, sodium lauryl sulfate and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

Solid compositions of a similar type can also be employed as fillers in soft-filled and hard-filled gelatin capsules using excipients such as lactose and high molecular weight polyethylene glycols and the like.

The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and may also be of a composition that they release the active ingredient(s) in a delayed manner only, or optionally preferentially, in a certain part of the intestinal tract. Examples of embedding compositions that can be used include polymeric substances and waxes.

Solid compositions of a similar type can also be employed as fillers in soft-filled and hard-filled gelatin capsules using excipients such as lactose and high molecular weight polyethylene glycols and the like.

Dosage forms for topical or transdermal administration of the pharmaceutical compositions of this invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The particles are mixed under sterile conditions with a pharmaceutically acceptable carrier and any desired preservatives or buffers that may be required. Ophthalmic formulations, ear drops, and eye drops are also encompassed within the scope of the present invention.

In certain embodiments, pharmaceutically acceptable topical formulations of the invention comprise at least one compound of the invention and a penetration enhancer. The choice of topical formulation will depend on several factors including the condition to be treated, the physicochemical characteristics of the compound of the invention and other excipients present, its stability in the formulation, available manufacturing equipment, and cost constraints. As used herein, the term "penetration enhancer" means an agent capable of transporting a pharmacologically active compound across the stratum corneum and into the epidermis or dermis, preferably with little or no systemic absorption. Various compounds have been evaluated for their utility in increasing the rate of drug penetration through the skin. See, e.g., Percutaneous Penetration Enhancers, Maibach H.I. and Smith H.E. (eds), CRC Press, Inc., Boca Raton, Fla. (1995), which investigates the use and testing of various skin penetration enhancers, and Buyuktimkin et al., Chemical Means of Transdermal Drug Permeation Enhancement in Transdermal and Topical Drug Delivery Systems, Gosh T.K., Pfister W.R., Yum S.I. (eds.), Interpharm Press Inc., Buffalo Grove, Ill. (1997). In certain exemplary embodiments, penetrants useful in the present invention include, but are not limited to, triglycerides (e.g., soybean oil), aloe compositions (e.g., aloe vera gel), ethanol, isopropanol, Octylphenyl macrogol, oleic acid, macrogol 400, propylene glycol, N-decylmethyl sulfoxide, fatty acid esters (e.g., isopropyl myristate, methyl laurate, glycerol monooleate ester and propylene glycol monooleate) and N-methylpyrrolidone.

Transdermal patches have the added advantage of providing controlled delivery of the compound to the body. Such dosage forms can be made by dissolving or dispersing microparticles or nanoparticles in the appropriate medium. Absorption enhancers can also be used to increase the amount of the compound transdermally. The rate can be controlled by providing a rate controlling membrane or dispersing the particles in a polymer matrix or gel.

In certain embodiments, the composition can be in the form of an ointment, paste, cream, lotion, gel, powder, solution, spray, inhalant, or patch. In certain exemplary embodiments, compositions of the invention are formulated as creams, which may additionally contain saturated or unsaturated fatty acids such as stearic acid, palmitic acid, oleic acid, palmityl-oleic acid, cetyl alcohol, or Oleyl alcohol, stearic acid is especially preferred. The creams of the present invention may also contain nonionic surfactants, such as polyoxy-40-stearate. In certain embodiments, the active ingredient is admixed under sterile conditions with a pharmaceutically acceptable excipient and any desired preservatives or buffers that may be required. Ophthalmic formulations, ear drops, and eye drops are also encompassed within the scope of the present invention. Furthermore, the present invention encompasses the use of transdermal patches, which have the added advantage of providing controlled delivery of compounds of the present invention to the body. Such dosage forms are made by dissolving or dispersing the compound in the proper medium. Penetration enhancers, as noted above, can also be used to increase the amount of the compound which penetrates the skin. The rate can be controlled by providing a rate controlling membrane or dispersing the compound in a polymer matrix (eg PLGA) or gel.

Ointments, pastes, creams, and gels may contain excipients such as animal and vegetable fats, oils, waxes, paraffins, starches, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicon acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons.

It is also understood that the compounds and pharmaceutical compositions of the present invention may be formulated and employed in combination therapy, that is, the compounds and pharmaceutical compositions may be formulated with one or more other desired treatments or medical procedures or with one or more Administered simultaneously with, before or after other desired treatments or medical procedures. The particular combination of therapies (therapeutics or procedures) to be used in a combination regimen will take into account the compatibility of the desired therapeutics and/or procedures and the desired therapeutic effect to be achieved. It is also understood that the therapy employed may achieve the desired effect for the same condition (for example, a provided compound may be administered concomitantly with another agent effective against beta-thalassemia or sickle cell anemia), or it may achieve a different effect (eg to control any adverse effects).

It is also to be understood that particular compounds of the invention may exist in free form, or, where appropriate, in the form of pharmaceutically acceptable derivatives thereof, to be used in therapy. According to the present invention, pharmaceutically acceptable derivatives include, but are not limited to, pharmaceutically acceptable salts, esters, salts of such esters, or prodrugs or other adducts or derivatives of the compounds of the present invention, which in Administration to a patient in need thereof can provide, directly or indirectly, a compound otherwise described herein, or a metabolite or residue thereof.

treatment method

The present invention provides compounds that induce gamma globulin and produce beneficial therapeutic effects. In certain embodiments, the compounds and compositions described herein are used to treat a hemoglobinopathy, such as sickle cell anemia or beta-thalassemia. In certain embodiments, provided compounds or compositions are used to treat sickle cell anemia. In certain other embodiments, provided compounds or compositions are used to treat beta-thalassemia.

The methods of the invention stimulate gamma globulin expression. In one embodiment, the present invention provides a method comprising a compound of the present invention or a composition thereof for stimulating gamma globulin expression, comprising: subjecting an individual to a compound or composition of the present invention in an environment suitable for inducing gamma globulin in the individual; contact under conditions of protein expression.

In some embodiments, the present invention provides a method for inducing gamma hemoglobulin, which comprises: allowing cells to react with an effective amount of formula (I-a), (II), (III), (IV), (V), (VI ) or (VII) compound contact. In certain embodiments, the present invention provides a method for inducing gamma hemoglobulin, comprising: administering to an individual an effective amount of formula (I-a), (II), (III), (IV), (V), ( VI) or (VII) compound.

In certain embodiments, the present invention provides methods of treating β-thalassemia, comprising: administering an effective amount of a provided compound or composition to a patient suffering from β-thalassemia.

In certain embodiments, the present invention provides methods of treating sickle cell anemia comprising: administering to a patient suffering from sickle cell anemia an effective amount of a provided compound or composition.

In certain embodiments, provided compounds or compositions are administered orally. In certain embodiments, provided compounds or compositions are administered parenterally. In certain embodiments, provided compounds or compositions are administered in combination with other therapeutic agents.

The compounds of the invention are preferably formulated in dosage unit form for ease of administration and uniformity of dosage. The expression "unit dosage form" as used herein refers to a physically discrete unit of therapeutic agent suitable for the patient to be treated. It should be understood, however, that the total daily usage of the compounds and compositions of the present invention will be determined by the attending physician within the scope of sound medical judgment. The particular therapeutically effective dose for any particular patient or organism will depend on a variety of factors including the condition being treated and the severity of the condition; the activity of the particular compound employed; the particular composition employed; the age, weight of the individual , general health, sex, and diet; time of administration, route of administration (mute), and rate of excretion of the particular compound used; duration of treatment; drugs used in combination or concomitantly with the particular compound used; similar factors well known in the medical art (see e.g. Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th Edition, eds. A. Gilman, J. Hardman and L. Limbird, McGraw-Bill Press, 155-173, 2001, the entire contents of which are incorporated herein by reference).

screening method

In one embodiment, the invention provides methods of identifying compounds that induce gamma globulin expression. In certain embodiments, the invention provides cell-based assays comprising incubating a test compound with MEL cells containing the human gamma globulin promoter linked to a first fluorescent reporter and linked to a second fluorescent reporter. the human beta hemoglobin promoter of the fluorescent reporter; and measuring the fluorescence intensity compared to the background signal. In certain embodiments, the dual fluorescent reporter plastid is pmLAR-Gp-DsRed-Bp-d2EGFP. In certain embodiments, the invention provides a MEL cell comprising a MEL cell comprising a human γ-hemoglobin promoter linked to a first fluorescent reporter and a human β-hemoglobin promoter linked to a second fluorescent reporter . In certain embodiments, the first fluorescent reporter is DsRed. In certain embodiments, the second fluorescent reporter is d2EGFP. In certain embodiments, the first fluorescent reporter is DsRed and the second fluorescent reporter is d2EGFP. In some embodiments, fluorescence intensity is measured in DsRed.

In certain embodiments, assays for determining the ability of a compound to induce gamma globulin expression are provided. In other embodiments, compounds of the invention exhibit _EC50 values < 100 μΜ. In other embodiments, compounds of the invention exhibit EC50 values < ₅₀ μΜ. In other embodiments, compounds of the invention exhibit _EC50 values < 40 μΜ. In other embodiments, compounds of the invention exhibit _EC50 values < 30 μΜ. In other embodiments, compounds of the invention exhibit _EC50 values < 20 μΜ. In other embodiments, compounds of the invention exhibit _EC50 values < 10 μΜ. In other embodiments, compounds of the invention exhibit _EC50 values < 7.5 μΜ. In certain embodiments, compounds of the invention exhibit _EC50 values < 5 μM. In other embodiments, compounds of the invention exhibit _EC50 values < 2.5 μΜ. In certain embodiments, compounds of the invention exhibit _EC50 values < 1 μM. In certain embodiments, compounds of the invention exhibit _EC50 values < 0.75 μΜ. In certain embodiments, compounds of the invention exhibit _EC50 values < 0.5 μΜ. In certain embodiments, compounds of the invention exhibit _EC50 values < 0.25 μΜ. In certain embodiments, compounds of the invention exhibit _EC50 values < 0.1 μΜ. In other embodiments, compounds of the invention exhibit _EC50 values < 75 nM. In other embodiments, compounds of the invention exhibit EC50 values < ₅₀ nM. In other embodiments, compounds of the invention exhibit _EC50 values < 25 nM. In other embodiments, compounds of the invention exhibit _EC50 values < 10 nM. In other embodiments, exemplary compounds exhibit _EC50 values < 5 nM. In other embodiments, exemplary compounds exhibit _EC50 values < 1 nM.

According to the present invention, compounds can be analyzed in any available assay known in the art to identify gamma globulin inducers. For example, assays can be in vivo or in vitro, or high-throughput or low-throughput formats.

These and other aspects of the invention will be further understood by considering the following examples, which are intended to illustrate certain specific embodiments of the invention, but are not intended to limit the scope thereof, which is defined by the claims.

Example

A dual fluorescent reporter assay system for screening potential HbF inducers has been established. Using the fluorescent signal detected by the fluorometer, these compounds can be quickly interrogated to determine which compounds switch on the fetal gamma globulin promoter. This dual fluorescent reporter system allowed us to identify genes with a common core structure (benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one) and / Several heterocyclic compounds with high potency/specificity for the fetal hemoglobin chain. These compounds can be developed into a new generation of therapeutic drugs for the cure of hemepathies including sickle cell anemia and β-thalassemia (eg β-thalassemia major).

Materials and methods

Compounds include compound A (8-(3-carboxy-7-oxo-7H-benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-4-yl)-naphthalene -1,4,5-tricarboxylic acid), compound B (benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one), compound C (3-chloro -Benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one), compound D (4-nitro-benzo[de]benzo[4,5 ]imidazo[2,1-a]isoquinolin-7-one), compound E (4-methylamino-benzo[de]benzo[4,5]imidazo[2,1-a]iso Quinolin-7-one) and compound F (7-oxo-7H-benzo[de]benzo[4,5]imidazo[2,1-a]isoquinoline-3,4-dicarboxylic acid) All heterocyclic compounds included in the screen were purchased from ChemDiv Co. The selective CaMKK inhibitor STO-609 (7-oxo-7H-benzoimidazo[2,1-a]benzo[de]isoquinoline-3-carboxylic acid acetate) was purchased from Sigma-Aldrich.

Reporter construct. The reporter plastid pmLAR-Gp-DsRed-Bp-d2EGFP was constructed by a multiple-step subcloning process. Briefly, an 8,003 bp fragment of mini-LAR excised from pLAR-β (mLAR; the minimal locus containing the HS4-HS3-HS2-HS1 cis-acting module for regulation of human β-like hemoglobin gene expression was activated region) was inserted into the XhoI and EcoRI sites of pd2EGFP-1. Next, a 1,622bp β-hemoglobin promoter (Bp) fragment was generated by PCR amplification and cloned into the AgeI site of pd2EGFP-1 to generate pmLAR-Bp-d2EGFP. A 1,377bp gamma globulin promoter (Gp) fragment was also subcloned into the AgeI site of the pDsRed-Monomer-C1 vector, resulting in pGp-DsRed-C1. Then the Gp-DsRed fragment was excised from the pGp-DsRed-C1 plastid and reinserted into the SalI site between the mLAR of pmLAR-Bp-d2EGFP and Bp-d2EGFP. The resulting reporter pmLAR-Gp-DsRed-Bp-d2EGFP was then used to form a stable cell line by transfection into MEL cells.

Cell cultures and stable cell lines forming mouse erythroleukemia (MEL) cells and adult erythroid cell lines (Friend et al., (1971) Proc Natl Acad Sci USA68:378-82) were maintained in supplemented with 10% FBS and 1% Penicillin-streptomycin in DMEM medium (Gibco) (in a chamber at 37° C., 5% CO ₂ and humid atmosphere). To form a stable cell line, the reporter pmLAR-Gp-DsRed-Bp-d2EGFP was transfected into MEL cells using the Neon ^™ transfection system (Invitrogen). 2 × 106 MEL cells were transfected with ⁵ μg of plastids. After microporation, MEL cells were seeded in 10 ml antibiotic-free DMEM for 24 hours and selected with 700 mg/ml neomycin for 1 month. Primary human erythroid cultures were initiated by maintaining purified monocytes in SFEM medium (StemSpan) with 1 x cc100 cytokine cocktail for 7 days. Cells were then re-maintained in differentiation medium (SFEM with 20 ng/ml SCF, 1 U/ml EPO, 5 ng/ml IL-3, 2 μmol dexamethasone) at a density of 0.1-1× ¹⁰ cells/ml ) for 7 days. Cells were treated with compound on day 7 post-differentiation and harvested for further analysis 3 days after compound treatment.

Machine automated screening of compounds. Inoculate 5×10 ⁴ MEL cells carrying dual fluorescent reporters on an optical Packard 96-well visual culture plate, treat with 10 mM individual compounds for 3 days, and use Wallac Victor31420 multi-label counter (Ex: 550/9; Em: 620) A primary screen was performed for its DsRed intensity. Using digital image detector, CellomicsArrayscan3.0 system for secondary screening, this double confirms the intracellular DsRed fluorescence induced by compound treatment. Endogenous hemoglobin gene expression patterns in primary and secondary screen positive MEL cells and compound-treated primary human erythroid cultures were subsequently identified by real-time RT-PCR analysis.

Real-time RT-PCR analysis Total RNA was isolated using the RNAspin Mini Kit (GE Healthcare). cDNA synthesis was performed using SuperScript II reverse transcriptase (Invitrogen). Quantitative RT-PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems) and ABI7500 real-time system. All data were analyzed after correction for the expression of the mouse or human β-actin gene.

result

Formation of a high-throughput screening system for investigating embryonic/fetal hemoglobin gene-inducing compounds.

For the purpose of high-throughput screening of chemicals that induce embryonic/fetal hemoglobin gene expression, a dual fluorescent reporter with built-in human γ-hemoglobin promoter and β-hemoglobin promoter has been constructed. Downstream of the recombination locus control region (mLAR), the order of the gamma and beta haemoglobin promoters is organized according to their parental arrangement in the chromosomal beta-like haemoglobin locus. The NCBI GenBank ID for the human beta hemoglobin locus is NG_000007. The plastid pmLAR-Gp-DsRed-Bp-d2EGFP carries DsRed and d2EGFP fluorescent genes, which are respectively used as reporters for monitoring the expression of γ and β hemoglobin genes ( FIG. 2A ). Fluorescent signals from reporters of endogenous hemoglobin expression patterns in erythroid cells at different developmental stages have been compared. The expression of the fluorescent signal really reflects the state of the expression mode of the corresponding endogenous hemoglobin gene in the MEL and K562 cells.

Subsequently, dual fluorescent reporter plastids were used for high-throughput screening of compounds (Fig. 2B). Briefly, 10,000 compounds were selected to be tested for their ability to increase the embryonic/fetal hemoglobin promoter at a working concentration of 10 μM. The enhanced fluorescence expression was detected by a fluorescence reader and a digital image detector. Screening results are shown in the bitmap in Figure 2C. Eight out of 10,000 compounds showed at least a 1.5-fold increase in red fluorescence intensity compared to background signal. These compounds induced activation of endogenous embryonic/fetal hemoglobin chains was further verified by real-time RT-PCR analysis. The data show that only one compound (Compound A; arrow in Figure 2C) induces more than 10-fold expression of mouse embryonic (εγ) hemoglobin mRNA in MEL cells.

The sequences of mLAR, γ-globulin promoter and β-globulin promoter are as follows:

mLAR (8003bp, SEQ ID NO: 1):

tcgactctagaggatcaattcgagctcttggggaccccagtacacaagaggggacgcagggtatatgtagacatctcattctttttcttagtgtgagaataagaatagccatgacctgagtttatagacaatgagcccttttctctctcccactcagcagctatgagatggcttgccctgcctctctactaggctgactcactccaaggcccagcaatgggcagggctctgtcagggctttgatagcactatctgcagagccagggccgagaaggggtggactccagagactctccctcccattcccgagcagggtttgcttatttatgcatttaaatgatatatttatlttaaaagaaataacaggagactgcccagccctggctgtgacatggaaactatgtagaatattttgggttccatttttttttccttctttcagttagaggaaaaggggctcactgcacatacactagacagaaagtcaggagctttgaatccaagcctgatcatttccatgtcatactgagaaagtccccacccttctctgagcctcagtttctctttttataagtaggagtctggagtaaatgatttccaatggctctcatttcaatacaaaatttccgtttattaaatgcatgagcttctgttactccaagactgagaaggaaattgaacctgagactcattgactggcaagatgtccccagaggctctcattcagcaataaaattctcaccttcacccaggcccactgagtgtcagatttgcatgcactagttcacgtgtgtaaaaaggaggatgcttctttcctttgtattctcacatacctttaggaaagaacttagcacccncccacacagccatcccaataactcatttcagtgactcaacccttgactttataaaagtcttgggcagtatagagcagagattaagagtacagatgctggagccagaccacctgagtgatcagtgactcagtttctcttagtaa ttgtatgactcagtttcttcatctgtaaaatggagggttttttaattagtttgtttttgagaaagggtctcactctgtcacccaaatgggagtgtagtggcaaaatctcggctcactgcaacttgcacttcccaggctcaagcggtcctcccacctcaacatcctgagtagctggaaccacaggtacacaccaccatacctcgctaattttttgtatttttggtagagatggggtttcacatgttacacaggatggtctcagactccggagctcagaagagtcaagcatttgcctaaggtcggacatgtcagaggcagtgccagacctatgtgagactctgcagctacgtctcatgggccctgtgctgcactgatgaggaggatcagatggatggggcaatgaagcaaaggaatcattctgtggataaaggagacagccatgaagaagtctatgactgtaaatttgggagcaggagtctctaaggacttggatttcaaggaattttgactcagcaaacacaagaccctcacggtgactttgcgagctggtgtgccagatgtgtctatcagaggttccagggagggtggggtggggtcagggctggccaccagctatcagggcccagatgggttataggctggcaggctcagataggtggtcaggtcaggctggtggtgctgggtggagtccatgactcccaggagccaggagagatagaccatgagtagagggcagacatgggaaaggtgggggaggcacagcatagcagcatttttcattctactactacatgggactgctcccctatacccccagctaggggcaagtgccttgactcctatgttttcaggatcatcatctaIaaagtaagagtaataattgtgtctatctcatagggttattacgaggatcaaaggagatgcacactctctggaccagtggcctaacagttcaggacagagctatgggcttcctatgtatgggtcagtggtctcaatg tagcaggcaagttccagaagatagcatcaaccactgctagagatatactgccagtctcagagcctgatgttaatttagcaatgggctgggaccctcctccagtagaaccttctaaccaggatccagtggggcctctaagactaagtcactctgtctcactgtgtcttagccagttccttacagcttgccctgatgggagatagagaatgggtatcctccaacaaaaaaataaattttcatttctcaaggtccaacttacgttttcttaatttttaaaaaaatcttgaccattctccactctctaaaataatccacagtgagagaaacattcttttcccccatcccataaatacctctattaaatatggaaaatctgggcatggtgtctcacacctgtaatcccagcactttgggaggctgaggtgggtggactgcttggagctcaggagttcaagaccatcttggacaacatggtgataccctgcctctacaaaaagtacaaaaattagcctggcatggtggtgtgcacctgtaatcccagctattagggtggctgaggcaggagaatcgcttgaacccgggaggcggaggttgcagtgagctgagatcgtgccactgcactccagcctgggggacagagcacattataattaactgttattttttacttggactcttgtggggaataagatacatgttttattcttatnatgattcaagcactgaaaatagtgtttagcatccagcaggtgcttcaaaaccatttgctgaatgattactatactttttacaagctcagctccctctatcccttccagcatcctcatctctgattaaataagcttcagtttttccttagttcctgttacatttctgtgtgtctccattaggtacctcccatagtccaagcatgagcagttctggccaggcccctgtcggggtcagtgccccacccccgccttctggttctgtgtaaccttctaagcaaaccttctggctc aagcacagcaatgctgagtcatgatgagtcatgctgaggcttagggtgtgtgcccagatgctctcagcctagagtgatgactcctatctgggtccccagcaggatgcttacagggcagatggcaaaaaaaaggagaagctgaccacctgactaaaactccacctcaaacggcatcataaagaaaatggatgcctgagacagaatgtgacatattctagaatatattatttcctgaatatatatatatatatatatacacatatacgtatatatatatatatatatatatttgttgttatcaattgccatagaatgattagttattgtgaatcaaatatttatcngcaggtggcctctatacctagaagcggcagaatcaggctttattaatacatgtgtatagatttttaggatctatacacatgtattaatatgaaacaaggatatggaagaggaaggcatgaaaacaggaaaagaaaacaaaccttgtttgccattttaaggcacccctggacagctaggtggcaaaaggcctgtgctgttagaggacacatgctcacatacggggtcagatcaattctgtctgangttctctgacttatctaccattttccctccttaaagaaactgtggaacttccttcagctagaggggcctggctcagaagcctctggtcagcatccaagaaatacttgatgtcactttggctaaaggtatgatgtgtagacaagctccagagatggtnctcatttccatatccacccacccagctttccaattttaaagccaattctgaggtagagactgtgatgaacaaacaccttgacaaaattcaacccaaagactcactngcctagcttcaaaatccttactctgacatatactcacagccagaaattagcatgcactagagtgtgcatgagtgcaacacacacacacaccaattccatattctctgtcagaaaatcctgttggtttttcgtgaaaggatgttttcaga ggctgaccccttgccttcacctccaatgctaccactctggtctaagtcactgtcaccaccacctaaattatagctgttgactcataacaatcttcctgcttctaccactgccccactacaatttcttcccaatatactatccaaattagtcttttcaaaatgtaagtcatatatggtcacctctttgttcaaagtcttctgatagtttcctatatcatttataataaaaccaaatccttacaattctctacaatagttgncatgcatatattatgmattacagatacgcatatatatagctctcatataaataaatatatatatttatgtgtatgtgtgtagagtgttttttcttacaactctatgatgtaggtattattagtgtcccaaattttataatttaggacttctatgatctcatcttttattctccccttcaccgaatctcatcctacattggccttattgatattccttgaaaattctaagcatcttacatctttagggtatnacatttgccattccctatgccctaaatatttaatcatagtttcatataaatgggttcctcatcatctatgggtactctctcaggtgtIaactttatagtgaggactttcctgccatactacttaaagtagcgataccctttcaccctgtcctaatcacactctggccttcatttcagttttttttttttctccatagcacctaatctcattggtatataacatgntcatttgcttatttaatgtcaagctctttccactatcaagtccatgaaaacaggaacmattcctctattctgtttttgtgctgtattcttagcaattttacaattttgaatgaaatgaatgagcagtcaaacacatatacaactataattaaaaggatgtatgctgacacatccactgctatgcacacacaaagaaatcagtggagtagagctggaagcgctaagcctgcatagagctagttagccctccgcaggcagagcctt gatgggattactgagttctagaattggactcatttgttttgtaggctgagatngctcttgaaaacttgttctgaccaaaataaaaggctcaaaagatgaatatcgaaaccagggtgttttttacactggaatttataactagagcactcatgtttatgtaagcaattaattgtttcatcagtcaggtaaaagtaaagaaaaactgtgccaaggcaggtagcctaatgcaatatgccactaaagtaaacattattccataggtgtcagatatggcttattcatccatcttcatgggaaggatggccttggcctggacatcagtgttatgtgaggttcaaaacacctctaggctataaggcaacagagctccttttttttttttctgtgctttcctggctgtccaaatctctaatgataagcatacttctattcaatgagaatattctgtaaganatagttaagaattgtgggagccattccgtctcttatagttaaatttgagcttcttttatgatcactgtttttttaatatgctttaagttctggggtacatgtgccatggtggtttgctgcacccatcaacccgtcatctacattaggtatttctcctaatgctatccttcccctagccccccacccccaacaggccccagtgtgtgatgttcccctccctgtgtccatggatcactggttttttttttttttttttttttttttttaaagtctcagttaaatttttggaatgtaatnatmcctggtatcctaggacctgcaagttatctggtcacmagccctcacgttttgatgataatcacatatttgtaaacacaacacacacacacacacacacacacatatatatatataaaacatatatatacataaacacacataacatatttatcgggcatttctgagcaactaactcatgcaggactctcaaacaccaacctatagccttttctatgtatctacttgtgtagaaaccaagcgtggggac tgagaaggcaatagcaggagcattctgactctcactgcctttggctaggtccctccctcatcacagctcagcatagtccgagctcttatctatatccacacacagtttctgacgctgcccagctatcaccatcccaagtctaaagaaaaaaataatgggtttgcccatctctgttgattagaaaacaaaacaaaataaaataagcccctaagctcccagaaaacatgactaaaccagcaagaagaagaaaatacaataggtatatgaggagactggtgacactagtgtctgaatgaggcttgagtacagaaaagaggctctagcagcatagtggtttagaggagatgtttctttccttcacagatgccttagcctcaataagcttgcggttgtggaagtttactttcagaacaaactcctgtggggctagaattattgatggctaaaagaagcccgggggagggaaaaatcattcagcatcctcacccttagtgacacaaaacagagggggcctggttttccatatttcctcatgatggatgatctcgttaatgaaggtggtctgacgagatcattgcttcttccatttaagccttgctcacttgccaatcctcagttttaaccttctccagagaaatacacattttttattcaggaaacatactatgttatagtttcaatactaaataatcaaagtactgaagatagcatgcataggcaagaaaaagtccttagctttatgttgctgttgtttcagaatttaaaaaagatcaccaagtcaaggacttctcagttctagcactagaggtggaatcttagcatataatcagaggtttttcaaaatttctagacatgagattcaaagccctgcacttaaaatagtctcatttgaattaactctttatataaattgaaagcacattctgaactacttcagagtangttttatttctatgttcttagttcataaatacattaggcaatgcaatttaattaaaaa aacccaagaatttcttagaattttaatcatgaaaataaatgaaggcatctttacttactcaaggtcccaaaaggtcaaagaaaccaggaaagtaaagctatatttcagcggaaaatgggatatttatgagttttctaagttgacagactcaagttnaaccttcagtgcccatgatgtaggaaagtgtggcataactggctgattctggctttctactcctttttcccattaaagatccctcctgcttaattaacattcacaagtaactctggttgtactnaggcacagtggctcccgaggtcagtcacacaataggatgtctgtgctccaagttgccagagagagagattactcttgagaatgagcctcagccctggctcaaactcacctgcaaacttcgtgagagatgaggcagaggtacactacgaaagcaacagttagaagctaaatgatgagaacacatggactcatagagggaaacaacgcatactggggcctatcagagggtggagggtgagagaaggagaggatcaggaaaaatcactaatggatgctaagcgcaatacctgagtgatgagatcatctatacaacaaacccccttgacattcatttatctatgtaacaaacctgcacatcctgtacacgtacccctgaacttaaaataaaagttgaaaacaagaaagcaacagtttgaacacttgttatggtctattctctcattctttacaattacactagaaaatagccacaggctcctgcaaggcagccacagaatttatgacttgtgatgatctaatgctttcataaagaagcaaatataataaatactataccacaaatgtaatgtttgatgtctgataatgatatttcagtgtaanaaacttagcactcctatgtatattatttgatgcaataaaaacatattttttttagcacttacagtctgccaauactggcctgtgacacaaaaaaagtttag

Gamma hemoglobin promoter (1377bp, SEQ ID NO: 2):

tacacaggatcatgaaggatgaaagaacttcaccaatatcataataatttcaatcaacctgatagcttaggggataaactaatttgaagatacagcttgcctccgataagccagaattccagagcttctggcatcataatccagcaaggccagagatcatggatcactttcagagaaaaacaaaaacaaactaaccaaaagcaaaacagaaccaaaaaaccaccataaatacttcctaccctgtcaatggtccaatatgtcagaaacagcgctgtgttagaaataaagctgcccaaagtacactaatacccgagttataatagtgtggactattagtcaataaaaacaaccctcgcctctttagagttgttttccatgtacacgcacatcttatgtctcagagtaagattccctgagaagtgaacctagcatttatacaagataatcaattctaatccacagtacctgccaaagaacattctaccatcatctttactgagcacagaaggctacgccaaaaccctgggtcatcagccagcacacacacttatccagtggtaaacacacatcatctggtgtatacatacatacctgaatatggaatcaaatatttttctaagatgaaacagtcatgatttatttcaaataggtacggataagtagatattgaggtaagcattaggtctcatattatgtaacaccaatccatcactgcgctgaaaccgtggcttt atagaaattgttttcaetgcaccattgagaaattaagagataatggcaaaagtcacaaagagtatattcaaaaagaagtatagcaetttttccttagaaaccactgctaactgaaagagactaagatttgtcccgtcaaaaatcctggacctatgcctaaaacacatttcacaatccctgaacttttcaaaaattggtacatgctttagctttaaactacaggcctcactggagctagagacaagaaggtaaaaaacggctgacaaa agaagtcctggtatcctctatgatgggagaaggaaactagctaaagggaagaataaattagagaaaaactggaatgactgaatcggaacaaggcaaaggctataaaaaaaattaagcagcagtatcctcttgggggccccttccccacactatctcaatgcaaatatctgtctgaaacggtccctggctaaactccacccatgggttggccagccttgccttgaccaatagccttgacaaggcaaacttgaccaatagtcttagagtatccagtgaggccaggggccggcggctggctagggatgaagaataaaaggaagcacccttcagcagttccacacactcgcttctggaacgtctgaggttatcaataagc

β-hemoglobin promoter (1622bp, SEQ ID NO: 3):

gtcgactctagaggatctctatttatttagcaataatagagaaagcatttaagagaataaagcaatggaaataagaaatttgtaaatttccttctgataactagaaatagaggatccagtttcttttggttaacctaaattttatttcattttattgttttattttattttattttattttattttgtgtaatcgtagtttcagagtgttagagctgaaaggaagaagtaggagaaacatgcaaagtaaaagtataacactttccttactaaaccgactgggtttccaggtaggggcaggattcaggatgactgacagggcccttagggaacactgagaccctacgctgacctcataaatgcttgctacctttgctguttaattacatcttttaatagcaggaagcagaactetgcacttcaaaagtttttcctcacctgaggagnaatttagtacaaggggaaaaagtacagggggatgggagaaaggcgatcacgttgggaagctatagagaaagaagagtaaattttagtaaaggaggtttaaacaaacaaaatataaagagaaataggaacttgaatcaaggaaatgattttaaaaegcagtattcttagtggactagaggaaaaaaataatctgagccaagtagaagaccttttcccctcctacccctactttctaagtcacagaggctttttgttececeagaeactcttgcagattagtccaggcagaaacagttagatgtccecagttaacctcctatttgacaccactgattaccccattgatagtcacactttgggttgtaagtgactttttatttatttgtatttttgactgcattaagaggtctctagttttttatctcttgtttcccaaaacctaataagtaactaatgcacagageacattgatttgtatttattctatttttagacataatttattagcatgcatgageaaattaagaaaaacaacaacaaatgaatgcatatat atgtatatgtatgtgtgtatatatacacatatatatatatattttttttcttttcttaccagaaggttttaatccaaataaggagaagatatgcttagaactgaggtagagttttcatccattctgtcctgtaagtattttgcatattctggagacgcaggaagagatccatctacatatcccaaagctgaattatggtagacaaagctcttccacttttagtgcatcaatttcttatttgtgtaataagaaaattgggaaaacgatcttcaatatgcttaccaagctgtgattccaaatattacgtaaatacacttgcaaaggaggatgtttttagtagcaatttgtactgatggtatggggccaagagatatatcttagagggagggctgagggtttgaagtccaactcctaagccagtgccagaagagccaaggacaggtacggctgtcatcacttagacctcaccctgtggagccacaccctagggttggccaatctactcecaggagcagggagggcaggagccagggctgggcataaaagtcagggcagagccatctattgcttacatttgctIctgacacaactgtgttcactagcaacctcaaacagacacc

Identification of compounds that enhance human embryonic/fetal eglobulin gene expression with greater potency and specificity.

Next, the induction of endogenous hemoglobin genes by Compound A in cultures of human erythroid progenitor cells was examined. Primary cultured cells were isolated from peripheral blood of donors, expanded and differentiated in culture medium for a total of 14 days, and treated with different concentrations of Compound A for an additional 3 days. Although the proliferation rate of cells was inversely related to the concentration of Compound A, the relative expression of γ (but not β) hemoglobin genes increased significantly with the concentration of Compound A ( FIG. 3A ).

To further investigate the unique feature of compound A that allows the induction of human embryonic/fetal hemoglobin genes in primary erythroid cultures, we first compared β-like hemocytes treated with different fetal hemoglobin inducers in primary erythroid cultures protein expression pattern. Interestingly, cells treated with compound A showed the highest fold induction of γ-hemoglobulin compared to cells treated with TSA, hydroxyurea (HU) and sodium butyrate (NaB), while compared to those observed with HU and TSA Compound A exhibited only minimal induction of the adult beta hemoglobin gene (Fig. 3B).

Identification of transcriptional regulators involved in the induction of fetal hemoglobin genes by Compound A treatment.

Genes including GATA1(18), NF-E2(3), EKLF(2), YY1(22), TR2/TR4(29), NF-E4(7), RREB1(4) and BCL11A(23) have been identified in Several transcription factors within the gene function as activators or repressors of hemoglobin gene expression. In order to reveal the molecular mechanism by which Compound A specifically induces the expression of the fetal γ-globin gene, the relative expression levels of several transcription factors known to affect the transcription of the gamma-globin gene were analyzed ( FIG. 4 ). It can be seen that the expression level of the γ-globulin gene activator NF-E4(7) is up-regulated in primary human erythroid cultures treated with compound A. At the same time, the expression level of the γ-hemoglobin gene suppressor Bcl11a (23) decreased. Compound A treatment did not significantly change the expression levels of other hemoglobin gene regulators (such as EKLF, GATA1, NF-E2, RREB1, CP2, TR2/TR4, YY1). Both the increase of NF-E4 and the decrease of Bcl11a may result in the activation of gamma globulin gene expression in these compound A-treated erythroid cells.

Compounds with the same heterocyclic core structure as compound A enhance fetal gamma globulin expression

Subsequently, 18 compounds having a heterocyclic structure similar to Compound A have been investigated. Five of these 18 heterocyclic compounds were identified to induce fetal gamma globulin expression (Figure 5, compounds B-F). Interestingly, all six compounds (including Compound A) share the same core structure (benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one) and The globulin inducibility is comparable. Figure 5 shows the dose effect of these heterocyclic compounds in activating gamma globulin gene expression and inhibiting cell proliferation.

The relative induction folds of the endogenous β and γ hemoglobin genes of compounds B, C and F at their _IC50 concentrations have been estimated by quantitative RT-PCR analysis and compared with the induction folds of compounds A, HU and NaB (Figure 6 ). To obtain a complete picture of these heterocyclic compounds regarding their inducibility to individual hemoglobin chains, the _IC50 values of HU, NaB or compounds A/B/C/F have also been estimated by absolute quantitative RT-PCR analysis. Absolute amount of mRNA encoding each hemoglobin in treated human erythroid cells. As seen in Figure 9, the proportion of γ-hemoglobin mRNA significantly increased from 8% to 10.7-19.6% after heterocyclic compound treatment. Meanwhile, the proportion of β-globulin mRNA decreased from 87.2% to 73.8-81.4% after heterocyclic compound treatment. Taken together, the identified heterocyclic compounds preferentially induced embryonic ε and fetal γ-globulin gene expression, but not adult β-globulin ( FIGS. 6 and 9 ).

Comparison of Heterocyclic Compounds with Butyric Acid or Hydroxyurea in Inducing Fetal Gamma Globulin

In order to evaluate the induction ability of these heterocyclic compounds on fetal hemoglobin gene compared with NaB and HU, the _IC50 value (half maximal inhibitory concentration) and EC value (effective concentration) of these gamma hemoglobin inducers have been calculated (Figure 10). In both, _IC50 is the concentration of compound that reduces cell proliferation by 50%. EC was defined as the concentration of the compound that induced γ-globulin mRNA content by 1.88-fold (ie, the average fold of γ-globulin gene induced by HU at _IC50 in three experiments). With different branching chains, these heterocyclic compounds show quite different cytotoxicity ( _IC50 ) as well as their effective dose (EC). The therapeutic window was then calculated as the ratio of the respective _IC50 and EC ( _IC50 /EC), which can be used to assess the benefit of these heterocycles against each other and compared to HU and NaB (4th row from left, Fig. 10). Considering the therapeutic advantage (therapeutic window), at least 3 of the identified compounds (Compounds B, C and E) showed therapeutic potential superior to HU or NaB. Furthermore, as seen in the rightmost row of the table in Figure 10, all 6 compounds showed similar or better ability to induce gamma hemoglobin gene expression at _IC50 compared to HU and NaB. In certain embodiments, compositions of the invention exhibit a greater therapeutic window than hydroxyurea. In certain embodiments, compositions of the invention exhibit a greater therapeutic window than sodium butyrate.

CaMKK inhibitor STO-609 increases the expression of fetal γ-globulin

Because STO-609(30), a selective inhibitor of Ca(2+)/calmodulin-dependent protein kinase (CaMKK), contains the same heterocyclic core structure as these fetal gamma hemoglobin-inducing compounds described above (benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one), so its potential to induce the expression of fetal gamma hemoglobin in human primary erythroid cells was next examined (Figure 7). As expected, after treatment with STO-609 at a concentration of 50 mM or above, the expression of fetal γ-globulin genes but not adult β-globulin genes was increased ( FIG. 7A ). As in the case of γ-globulin-induced heterocycles A-F, treatment with STO-609 also caused transcriptional upregulation of the γ-globulin gene activator NF-E4 (Fig. 7B). At the same time, STO-609 down-regulated the expression of γ-hemoglobin gene suppressor Bcl11a (Fig. 7C). In addition, it was found that STO-609 treatment also significantly reduced the transcript of c-Myb, another γ-hemoglobin gene suppressor (10) (Fig. 7D). Taken together, the data show that heterocyclic compounds, including the specific CaMKK inhibitor STO-609, which share the same core structure (benzo[de]benzo[ 4,5]imidazo-[2,1-a]isoquinolin-7-one). Activation of gamma globulin genes by these compounds appears to be stimulated in part by modulating the expression of certain gamma globulin gene regulators such as NF-E4, Bcl11a and/or cMyb. The possible role of these heterocyclic compounds, including A-F and STO609, and their crosstalk with the fetal gamma-globulin gene induction program is discussed below.

discuss

A high-throughput screening system using dual fluorescent reporter plastids as indicators for monitoring endogenous hemoglobin gene expression has been described above. A similar method has been reported and is also well characterized for its use in assessing the expression of endogenous hemoglobin genes (27). The specificity of these assays has been demonstrated during screening for HbF inducers. Using this dual fluorescence analysis system, it is possible to screen compounds that can induce the expression of gamma hemoglobin gene in adult erythroid cells. After surveying 10,018 compounds, six heterocyclic compounds have been identified, all comprising the same core structure (benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one ), which exhibited similar or better gamma-globulin-inducing efficacy than HU or NaB at the concentration of IC ₅₀ . Although these compounds exhibited varying degrees of cytotoxicity, all six heterocyclic compounds (including Compound A) were able to induce γ-globulin gene activation by 1.9 to 3.4 fold at IC ₅₀ ( FIG. 10 ). Furthermore, these heterocyclic compounds all exhibited a higher therapeutic window (IC ₅₀ /EC) than hydroxyurea, eg Compound B up to about 10-fold.

Fetal gamma globulin chains can functionally replace defective beta globulin chains and help prevent HbS formation in sickle cell anemia. The presence of fetal gamma hemoglobin chains also balances overloaded hemoglobin chains and prevents their precipitation in β-thalassemia patients. Therefore, increasing the amount of gamma hemoglobulin will reduce hemolysis in patients with hemoglobinopathy and effectively alleviate the severity of anemia. A cut-off value of 10%-20% HbF expression is considered effective in modulating the clinical severity of hemopathies (21). As demonstrated in Figure 9, Compounds A, B, C and F induced gamma hemoglobin expression ranging from 10.7-19.6% in primary erythroid cell cultures, indicating that their HbF inducing potency is comparable to that of other well-known fetal hemoglobin inducers ( For example, HU and NaB) are equivalent.

Because the six identified heterocyclic compounds and the specific CaMKK inhibitor STO-609 all share a common core structure, that is, the structure of compound B (benzo[de]benzo[4,5]imidazo[2,1- a] isoquinolin-7-one), it is expected that the naphthoylbenzimidazole skeleton is very likely to be the key to up-regulate/induce the expression of γ-globulin gene. In addition, it has been observed that the branched side chains of these compounds are located at position 3 or position 4 of the heterocyclic structure (as shown in Figure 5B) and there are variations in the potency and cytotoxicity of these compounds. Several compounds containing the same core structure (benzo[de]benzo-[4,5]imidazo[2,1-a]isoquinolin-7-one) but with branched chain modifications at other positions did not demonstrate possible Detected γ-globulin gene inducibility (Table 1). These data suggest that modification of certain positions, such as positions 3 and 4 of Compound B, may enable even better pharmaceutical development of compounds for sickle cell anemia and severe β-thalassemia major therapy.

Table 1. Screening compounds that did not exhibit detectable gamma globulin induction

Interestingly, STO-609, a specific CaMKK inhibitor with the same core structure as heterocyclic compounds A-F, also exhibited fetal γ-globulin inducibility. Inhibition of the cellular MEK/Erk pathway has also been shown to stimulate gamma globulin expression in human primary erythroid cells (14). Interestingly, CaMKK signaling is linked to the MEK/Erk pathway via CaMKI, a downstream target of CaMKK (25). Therefore, it is believed that inhibition of CaMKK activation by STO-609 would also trigger inhibition of MEK/Erk signaling, thus leading to elevated fetal gamma hemoglobin gene expression. In the case of the same core structure (benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one), the identified heterocyclic compounds A-F exhibit similarities with STO- 609 Similar capabilities, such as induction of fetal gamma hemoglobin, upregulation of NF-E4 mRNA, and downregulation of Bcl11a transcripts (and cMyb mRNA). As with CAMKK inhibitors, induction of fetal gamma-globulin expression by these heterocyclic compounds may be coupled in part to inhibition of MEK/Erk signaling.

In summary, studies have revealed that a series of heterocyclic compounds with a common core structure (benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one) can specifically induce Fetal gamma globulin expression in adult human erythroid cells. Furthermore, the modes of expression of several gamma-globulin regulators are elucidated and herein a description is presented of the mechanism describing how these heterocyclic compounds, including the specific CaMKK inhibitor STO-609, activate fetal gamma-globulin expression (Fig. 8). We propose a model in which these heterocyclic compounds stimulate the expression of fetal γ-globulin chains by regulating the expression of NF-E4, Bcl11a, c-Myb and/or interfering with MEK/Erk signaling. It has been well documented that overexpression of NF-E4 (32), downregulation of Bcl11a (23) and inhibition of MEK/Erk signaling pathway (14) induce endogenous fetal gamma hemoglobin expression. On the other hand, it was shown that overexpression of ectopic c-Myb suppresses fetal gamma globulin gene expression (10). Therefore, it is believed that these compounds and their derivatives with appropriate modifications are very valuable for the development of new generation drugs for curing hemopathies, especially sickle cell anemia and severe β-thalassemia.

references

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Claims (48)

1. a pharmaceutical composition, its contained (I-a) compound:

Or its pharmaceutically acceptable salt,

Wherein:

R¹、R²、R³、R⁴、R^6aAnd R^6bIt is each independently selected from hydrogen, halogen, alkyl, thiazolinyl, alkynyl, heterocyclic radical, aryl ,-OR^A、- OC(O)R^A、-N(R^B)₂、-N(R^A)C(O)R^A、-C(O)N(R^B)₂、-CN、-NO₂、-C(O)R^A、-C(O)OR^A、-SO₂R^AAnd- NHSO₂R^B；

Each R⁵Independently selected from hydrogen, halogen, alkyl, thiazolinyl, alkynyl, heterocyclic radical, aryl ,-OR^A、-OC(O)R^A、-N(R^B)₂、-N (R^A)C(O)R^A、-C(O)N(R^B)₂、-CN、-NO₂、-C(O)R^A、-C(O)OR^A、-SO₂R^AAnd-NHSO₂R^B；

Premise is R¹、R²、R³And R⁴One of person there is following formula:

Wherein R¹²And R¹³Independently selected from hydrogen ,-C (O) OR^A、-C(O)N(R^B)₂,-OH ,-NO2 ,-CN, halogen, alkyl, aryl and Heterocyclic radical, or R¹²And R¹³Carbocyclic ring or heterocycle is formed together with mediate atom；And

R¹⁴For hydrogen ,-C (O) OR^A、-C(O)N(R^B)₂、-OH、-NO₂,-CN, halogen, alkyl, aryl or heterocyclic radical；

Each R^AIndependently selected from hydrogen, alkyl, thiazolinyl, alkynyl, heterocyclic radical and aryl；

Each R^BIndependently selected from hydrogen, alkyl, thiazolinyl, alkynyl, heterocyclic radical and aryl, or two R^BShape together with mediate nitrogen Become heterocycle；

N is 0,1,2,3 or 4；And

Pharmaceutically acceptable excipient；

Wherein:

Each alkyl independently be substituted or be unsubstituted, straight chain or branched chain, non-annularity or ring-type saturated hydrocarbons, each thiazolinyl is only On the spot for containing being substituted or be unsubstituted of at least one double bond, straight chain or branched chain, non-annularity or ring-type unsaturated hydrocarbons, and Each alkynyl independently be containing being substituted or be unsubstituted of at least one three key, straight chain or branched chain, non-annularity or ring-type not Saturated hydrocarbons, wherein alkyl, thiazolinyl and alkynyl contain 1-10 carbon atom independently；

Each aryl independently be and is substituted or is unsubstituted phenyl, biphenyl, 1,2,3,4-tetralyl, naphthyl, anthryl or phenanthryl；

Each heterocyclic radical or heterocycle independently be and be substituted or be unsubstituted 3-to 10-ring, its contain 1 to 3 selected from oxygen, sulfur, Nitrogen or the hetero atom of phosphorus, remaining annular atoms is carbon atom；

Each carbocyclic ring independently be and is substituted or is unsubstituted cyclopenta, cyclohexyl, suberyl or aryl；

It is substituted and refers to through replacing selected from following one or more substituent groups: aliphatic group；Miscellaneous aliphatic group；Aryl；Heteroaryl；Virtue Base alkyl；Heteroaryl alkyl；Alkoxyl；Aryloxy；Miscellaneous alkoxyl；Heteroaryl epoxide；Alkyl sulfenyl；Artyl sulfo；Miscellaneous alkane Base sulfenyl；Heteroarylthio；-F；-Cl；-Br；-I；-OH；-NO₂；-CN；-CF₃；-CH₂CF₃；-CHCl₂；-CH₂OH；- CH₂CH₂OH；-CH₂NH₂；-CH₂SO₂CH₃；-C(O)R_x；-CO₂(R_x)；-CON(R_x)₂；-OC(O)R_x；-OCO₂R_x；-OCON (R_x)₂；-N(R_x)₂；-S(O)₂R_x；With-NR_x(CO)R_x, the most each R_xIndependently be aliphatic group, miscellaneous aliphatic group, aryl, heteroaryl, Aryl alkyl or heteroaryl alkyl, and wherein said aliphatic group；Miscellaneous aliphatic group；Aryl；Heteroaryl；Aryl alkyl；Heteroaryl alkane Base；Alkoxyl；Aryloxy；Miscellaneous alkoxyl；Heteroaryl epoxide；Alkyl sulfenyl；Artyl sulfo；Miscellaneous alkyl sulfenyl；Heteroaryl sulfur Base substituent group is without being further substituted with；

Wherein:

Each aliphatic group independently be alkyl, thiazolinyl, alkynyl or carbocyclic ring；

Each miscellaneous aliphatic group independently be containing one or more displacement oxygen of carbon atom, sulfur, nitrogen, phosphorus or the aliphatic group of silicon atom；

Each miscellaneous alkyl or miscellaneous alkyl-independently be containing one or more displacement oxygen of carbon atom, sulfur, nitrogen, phosphorus or the alkane of silicon atom Base；

Each heteroaryl independently be the cyclophane base with 5-10 annular atoms, and one of them annular atoms is selected from S, O and N；0,1 or 2 annular atomses are other hetero atoms independently selected from S, O and N；And remaining annular atoms is carbon；

Each group being connected with suffix-alkyl below refers to that this group is connected with core molecule part by alkyl；

Each group being connected with suffix-epoxide below refers to that this group is connected with core molecule part by oxygen atom；With

Each group being connected with suffix-sulfenyl below refers to that this group is connected with core molecule part by sulphur atom.

2. pharmaceutical composition as claimed in claim 1, wherein this compound has formula (II),

Or its pharmaceutically acceptable salt.

3. such as the pharmaceutical composition of claim 1 or 2, wherein R¹、R²、R³、R⁴And R⁵Be each independently selected from hydrogen ,-OH ,-Cl ,- Br、-F、C_1-6Alkyl, C_1-6Alkoxyl, alkynyl, aryl ,-NO₂、-N(R^B)₂、-C(O)CH₃、-C(O)OR^A、-C(O)N(R^B)₂、- CN, heterocyclic radical ,-SO₂-alkyl and-SO₂-aryl.

4. pharmaceutical composition as claimed in claim 3, wherein R¹、R²、R³、R⁴And R⁵Be each independently selected from hydrogen ,-OH ,-Cl ,-Br ,- F, methyl, ethyl, methoxyl group, ethyoxyl, phenyl, naphthyl ,-NO₂、-NH-C_1-6Alkyl ,-C (O) CH₃、-CO₂H、-CO₂Et、- CONH-aryl ,-CN, N-morpholinyl ,-SO₂-alkyl and-SO₂-aryl.

5. such as the pharmaceutical composition of claim 1 or 2, wherein R¹、R²、R³And R⁴In at least both be hydrogen.

6. pharmaceutical composition as claimed in claim 5, wherein R¹And R⁴For hydrogen.

7. pharmaceutical composition as claimed in claim 5, wherein R³And R⁴For hydrogen.

8. pharmaceutical composition as claimed in claim 5, wherein R¹And R²For hydrogen.

9. such as the pharmaceutical composition of claim 1 or 2, wherein R¹、R²、R³And R⁴In at least both are hydrogen, and R⁵For hydrogen.

10., such as the pharmaceutical composition of claim 1 or 2, wherein this compound has a formula (III), (IV) or (V):

Or its pharmaceutically acceptable salt.

11. such as claim 1 or the pharmaceutical composition of 2, wherein R¹²And R¹³Independently selected from hydrogen ,-C (O) OH ,-C (O) N (R^B)₂、-OH、-NO₂,-CN, halogen, alkyl, aryl and heterocyclic radical, or R¹²And R¹³Carbon is formed together with mediate atom Ring or heterocycle.

12. such as claim 1 or the pharmaceutical composition of 2, wherein R¹⁴For hydrogen ,-C (O) OH ,-C (O) N (R^B)₂、-OH、-NO₂、-CN、 Halogen, alkyl, aryl or heterocyclic radical.

13. such as claim 1 or the pharmaceutical composition of 2, wherein R²There is following formula:

The pharmaceutical composition of 14. such as claim 13, wherein R¹²And R¹³It is respectively-CO₂H。

The pharmaceutical composition of 15. such as claim 13, wherein R¹²、R¹³And R¹⁴It is respectively-CO₂H。

16. pharmaceutical compositions as claimed in claim 1, wherein R¹、R²、R³And R⁴One of person there is following formula:

Wherein R¹⁴Selected from hydrogen ,-C (O) OR^A、-C(O)N(R^B)₂、-OH、-NO₂,-CN, halogen, alkyl, aryl and heterocyclic radical；And R¹⁵ Selected from hydrogen, alkyl, aryl and heterocyclic radical.

The pharmaceutical composition of 17. such as claim 16, wherein R¹⁴For-CO₂H。

18. pharmaceutical compositions, it comprises and has formula (VI) or the compound of (VII):

Or its pharmaceutically acceptable salt,

Wherein:

R¹And R³It is each independently selected from hydrogen, halogen, alkyl, thiazolinyl, alkynyl, heterocyclic radical, aryl ,-OR^A、-OC(O)R^A、-N (R^B)₂、-N(R^A)C(O)R^A、-C(O)N(R^B)₂、-CN、-NO₂、-C(O)R^A、-C(O)OR^A、-SO₂R^AWith-NHSO₂R^B；

R⁵Selected from hydrogen, halogen, alkyl, thiazolinyl, alkynyl, heterocyclic radical, aryl ,-OR^A、-OC(O)R^A、-N(R^B)₂、-N(R^A)C(O) R^A、-C(O)N(R^B)₂、-CN、-NO₂、-C(O)R^A、-C(O)OR^A、-SO₂R^AWith-NHSO₂R^B；

R¹²、R¹³And R¹⁴It is each independently selected from hydrogen, halogen, alkyl, thiazolinyl, alkynyl, heterocyclic radical, aryl ,-OR^A、-OC(O)R^A、- SR^A、-N(R^B)₂、-N(R^A)C(O)R^A、-C(O)N(R^B)₂、-CN、-NO₂、-C(O)R^A、-C(O)OR^A、-S(O)R^A、-SO₂R^A、- SO₂N(R^B)₂And-NHSO₂R^B；Or R¹²And R¹³Carbocyclic ring or heterocycle is formed together with mediate atom；

Each R^AIndependently selected from hydrogen, alkyl, thiazolinyl, alkynyl, heterocyclic radical and aryl；And

Each R^BIndependently selected from hydrogen, alkyl, thiazolinyl, alkynyl, heterocyclic radical and aryl, or two R^BShape together with mediate nitrogen Become heterocycle；With

Pharmaceutically acceptable excipient；

Wherein:

Each alkyl independently be substituted or be unsubstituted, straight chain or branched chain, non-annularity or ring-type saturated hydrocarbons, each thiazolinyl is only On the spot for containing being substituted or be unsubstituted of at least one double bond, straight chain or branched chain, non-annularity or ring-type unsaturated hydrocarbons, and Each alkynyl independently be containing being substituted or be unsubstituted of at least one three key, straight chain or branched chain, non-annularity or ring-type not Saturated hydrocarbons, wherein alkyl, thiazolinyl and alkynyl contain 1-10 carbon atom independently；

Each aryl independently be and is substituted or is unsubstituted phenyl, biphenyl, 1,2,3,4-tetralyl, naphthyl, anthryl or phenanthryl；

Each heterocyclic radical or heterocycle independently be and be substituted or be unsubstituted 3-to 10-ring, its contain 1 to 3 selected from oxygen, sulfur, Nitrogen or the hetero atom of phosphorus, remaining annular atoms is carbon atom；

Each carbocyclic ring independently be and is substituted or is unsubstituted cyclopenta, cyclohexyl, suberyl or aryl；

It is substituted and refers to through replacing selected from following one or more substituent groups: aliphatic group；Miscellaneous aliphatic group；Aryl；Heteroaryl；Virtue Base alkyl；Heteroaryl alkyl；Alkoxyl；Aryloxy；Miscellaneous alkoxyl；Heteroaryl epoxide；Alkyl sulfenyl；Artyl sulfo；Miscellaneous alkane Base sulfenyl；Heteroarylthio；-F；-Cl；-Br；-I；-OH；-NO₂；-CN；-CF₃；-CH₂CF₃；-CHCl₂；-CH₂OH；- CH₂CH₂OH；-CH₂NH₂；-CH₂SO₂CH₃；-C(O)R_x；-CO₂(R_x)；-CON(R_x)₂；-OC(O)R_x；-OCO₂R_x；-OCON (R_x)₂；-N(R_x)₂；-S(O)₂R_x；With-NR_x(CO)R_x, the most each R_xIndependently be aliphatic group, miscellaneous aliphatic group, aryl, heteroaryl, Aryl alkyl or heteroaryl alkyl, and wherein said aliphatic group；Miscellaneous aliphatic group；Aryl；Heteroaryl；Aryl alkyl；Heteroaryl alkane Base；Alkoxyl；Aryloxy；Miscellaneous alkoxyl；Heteroaryl epoxide；Alkyl sulfenyl；Artyl sulfo；Miscellaneous alkyl sulfenyl；Heteroaryl sulfur Base substituent group is without being further substituted with；

Wherein:

Each aliphatic group independently be alkyl, thiazolinyl, alkynyl or carbocyclic ring；

Each miscellaneous aliphatic group independently be containing one or more displacement oxygen of carbon atom, sulfur, nitrogen, phosphorus or the aliphatic group of silicon atom；

Each miscellaneous alkyl or miscellaneous alkyl-independently be containing one or more displacement oxygen of carbon atom, sulfur, nitrogen, phosphorus or the alkane of silicon atom Base；

Each heteroaryl independently be the cyclophane base with 5-10 annular atoms, and one of them annular atoms is selected from S, O and N；0,1 or 2 annular atomses are other hetero atoms independently selected from S, O and N；And remaining annular atoms is carbon；

Each group being connected with suffix-alkyl below refers to that this group is connected with core molecule part by alkyl；

Each group being connected with suffix-epoxide below refers to that this group is connected with core molecule part by oxygen atom；With

Each group being connected with suffix-sulfenyl below refers to that this group is connected with core molecule part by sulphur atom.

The pharmaceutical composition of 19. such as claim 18, wherein R¹、R³、R⁵、R¹²、R¹³And R¹⁴Be each independently selected from-H ,-OH ,- Cl、-Br、-F、C_1-6Alkyl, C_1-6Alkoxyl, alkynyl, aryl ,-NO₂、-N(R^B)₂、-C(O)CH₃、-C(O)OR^A、-C(O)N (R^B)₂,-CN, heterocyclic radical ,-SO₂-alkyl and-SO₂-aryl.

The pharmaceutical composition of 20. such as claim 19, wherein R¹、R³、R⁵、R¹²、R¹³And R¹⁴Be each independently selected from-H ,-OH ,- Cl ,-Br ,-F, methyl, ethyl, methoxyl group, ethyoxyl, phenyl, naphthyl ,-NO₂、-NH-C_1-6Alkyl ,-C (O) CH₃、-CO₂H、- CO₂Et ,-CONH-aryl ,-CN, N-morpholinyl ,-SO₂-alkyl and-SO₂-aryl.

The pharmaceutical composition of 21. such as claim 18, wherein R¹、R³、R⁵、R¹²、R¹³And R¹⁴It is each independently selected from hydrogen ,-C (O) OR^A、-C(O)N(R^B)₂、-OH、-NO₂,-CN, halogen, alkyl, aryl and heterocyclic radical.

The pharmaceutical composition of 22. such as claim 21, wherein R¹、R³、R⁵、R¹²、R¹³And R¹⁴It is each independently selected from hydrogen ,-CO₂H、- C(O)N(R^B)₂、-OH、-NO₂,-CN, halogen, alkyl, aryl and heterocyclic radical.

23. pharmaceutical composition as any one of claim 18-22, wherein R¹²And R¹³Formed together with mediate atom Carbocyclic ring or heterocycle.

The pharmaceutical composition of 24. such as claim 23, wherein R¹²And R¹³Succimide is formed together with mediate atom Basic ring.

25. pharmaceutical composition as any one of claim 18-22, wherein R¹²And R¹³It is respectively-CO₂H。

26. pharmaceutical composition as any one of claim 18-22, wherein R¹²、R¹³And R¹⁴It is respectively-CO₂H。

27. pharmaceutical composition as any one of claim 18-22, wherein R³、R¹²、R¹³And R¹⁴It is respectively-CO₂H。

28. pharmaceutical composition as any one of claim 18-22, wherein R¹、R¹²And R¹³It is respectively-CO₂H。

29. pharmaceutical composition as any one of claim 18-22, wherein R¹、R¹²、R¹³And R¹⁴It is respectively-CO₂H。

30. pharmaceutical composition as any one of claim 1-2 and 18-22, wherein R⁵For hydrogen.

31. pharmaceutical composition as any one of claim 1-2, wherein R²It is not-C (O) R^A。

32. pharmaceutical composition as any one of claim 1-2, wherein R²It is not-C (O) CH₃。

33. pharmaceutical composition as any one of claim 1-2 and 18-22, wherein R³It it is not-NH-pi-allyl.

34. pharmaceutical composition as any one of claim 1-2, wherein R²It is not-NHCH₂CH₂OH。

35. pharmaceutical composition as any one of claim 1-2 and 18-22, wherein R¹And R⁴It is not-NO₂。

36. pharmaceutical compositions as any one of claim 1-2 and 18-22, wherein when n is 2, each R⁵It it is asynchronously methyl.

37. pharmaceutical compositions as claimed in claim 1, wherein this compound is:

Or its pharmaceutically acceptable salt.

38. pharmaceutical compositions as any one of claim 1-2,18-22 and 37, wherein said composition is selected from capsule, sheet The solid dosage forms for oral administration of agent, lozenge, pill, powder and granule.

39. the pharmaceutical composition as any one of claim 1-2,18-22 and 37, wherein said composition is selected from emulsion, micro- The liquid formulation for oral administration of emulsion, solution, suspension, syrup and elixir.

40. pharmaceutical compositions as any one of claim 1-2,18-22 and 37, wherein said composition is that sterile injectable is molten Liquid, suspension or emulsion.

41. pharmaceutical compositions are used for the purposes induced in the medicine of γ hyperglobulinemia in preparation, comprising:

Make the pharmaceutical composition thereof of cell and effective dose；

Wherein said pharmaceutical composition contained (I-a) compound:

Or its pharmaceutically acceptable salt,

Wherein:

R¹、R²、R³、R⁴、R^6aAnd R^6bIt is each independently selected from hydrogen, halogen, alkyl, thiazolinyl, alkynyl, heterocyclic radical, aryl ,-OR^A、- OC(O)R^A、-SR^A、-N(R^B)₂、-N(R^A)C(O)R^A、-C(O)N(R^B)₂、-CN、-NO₂、-C(O)R^A、-C(O)OR^A、-S(O)R^A、- SO₂R^A、-SO₂N(R^B)₂And-NHSO₂R^B；

Each R⁵Independently selected from hydrogen, halogen, alkyl, thiazolinyl, alkynyl, heterocyclic radical, aryl ,-OR^A、-OC(O)R^A、-SR^A、-N (R^B)₂、-N(R^A)C(O)R^A、-C(O)N(R^B)₂、-CN、-NO₂、-C(O)R^A、-C(O)OR^A、-S(O)R^A、-SO₂R^A、-SO₂N(R^B)₂ And-NHSO₂R^B；

Each R^AIndependently selected from hydrogen, alkyl, thiazolinyl, alkynyl, heterocyclic radical and aryl；

Each R^BIndependently selected from hydrogen, alkyl, thiazolinyl, alkynyl, heterocyclic radical and aryl, or two R^BShape together with mediate nitrogen Become heterocycle；And

N is 0,1,2,3 or 4；And

Pharmaceutically acceptable excipient；

Wherein:

Each alkyl independently be substituted or be unsubstituted, straight chain or branched chain, non-annularity or ring-type saturated hydrocarbons, each thiazolinyl is only On the spot for containing being substituted or be unsubstituted of at least one double bond, straight chain or branched chain, non-annularity or ring-type unsaturated hydrocarbons, and Each alkynyl independently be containing being substituted or be unsubstituted of at least one three key, straight chain or branched chain, non-annularity or ring-type not Saturated hydrocarbons, wherein alkyl, thiazolinyl and alkynyl contain 1-10 carbon atom independently；

Each aryl independently be and is substituted or is unsubstituted phenyl, biphenyl, 1,2,3,4-tetralyl, naphthyl, anthryl or phenanthryl；

Each heterocyclic radical or heterocycle independently be and be substituted or be unsubstituted 3-to 10-ring, its contain 1 to 3 selected from oxygen, sulfur, Nitrogen or the hetero atom of phosphorus, remaining annular atoms is carbon atom；

Each carbocyclic ring independently be and is substituted or is unsubstituted cyclopenta, cyclohexyl, suberyl or aryl；

It is substituted and refers to through replacing selected from following one or more substituent groups: aliphatic group；Miscellaneous aliphatic group；Aryl；Heteroaryl；Virtue Base alkyl；Heteroaryl alkyl；Alkoxyl；Aryloxy；Miscellaneous alkoxyl；Heteroaryl epoxide；Alkyl sulfenyl；Artyl sulfo；Miscellaneous alkane Base sulfenyl；Heteroarylthio；-F；-Cl；-Br；-I；-OH；-NO₂；-CN；-CF₃；-CH₂CF₃；-CHCl₂；-CH₂OH；- CH₂CH₂OH；-CH₂NH₂；-CH₂SO₂CH₃；-C(O)R_x；-CO₂(R_x)；-CON(R_x)₂；-OC(O)R_x；-OCO₂R_x；-OCON (R_x)₂；-N(R_x)₂；-S(O)₂R_x；With-NR_x(CO)R_x, the most each R_xIndependently be aliphatic group, miscellaneous aliphatic group, aryl, heteroaryl, Aryl alkyl or heteroaryl alkyl, and wherein said aliphatic group；Miscellaneous aliphatic group；Aryl；Heteroaryl；Aryl alkyl；Heteroaryl alkane Base；Alkoxyl；Aryloxy；Miscellaneous alkoxyl；Heteroaryl epoxide；Alkyl sulfenyl；Artyl sulfo；Miscellaneous alkyl sulfenyl；Heteroaryl sulfur Base substituent group is without being further substituted with；

Wherein:

Each aliphatic group independently be alkyl, thiazolinyl, alkynyl or carbocyclic ring；

Each miscellaneous aliphatic group independently be containing one or more displacement oxygen of carbon atom, sulfur, nitrogen, phosphorus or the aliphatic group of silicon atom；

Each miscellaneous alkyl or miscellaneous alkyl-independently be containing one or more displacement oxygen of carbon atom, sulfur, nitrogen, phosphorus or the alkane of silicon atom Base；

Each heteroaryl independently be the cyclophane base with 5-10 annular atoms, and one of them annular atoms is selected from S, O and N；0,1 or 2 annular atomses are other hetero atoms independently selected from S, O and N；And remaining annular atoms is carbon；

Each group being connected with suffix-alkyl below refers to that this group is connected with core molecule part by alkyl；

Each group being connected with suffix-epoxide below refers to that this group is connected with core molecule part by oxygen atom；With

Each group being connected with suffix-sulfenyl below refers to that this group is connected with core molecule part by sulphur atom.

Pharmaceutical composition any one of 42. claim 1-2,18-22 and 37 is used for treating β-mediterranean anemia in preparation Medicine in purposes, comprising:

The described pharmaceutical composition of effective dosage is to the patient suffering from β-mediterranean anemia.

It is lean that pharmaceutical composition any one of 43. claim 1-2,18-22 and 37 is used for treating sickle-cell anaemia type in preparation Purposes in the medicine of blood, comprising:

The described pharmaceutical composition of effective dosage is to the patient suffering from sickle-cell anaemia type anemia.

44. such as claim 42 or the purposes of 43, wherein said composition oral administration.

45. such as claim 42 or the purposes of 43, wherein said composition parenteral.

46. such as claim 42 or the purposes of 43, wherein said composition is administered with another therapeutic combination.

47. such as claim 42 or the purposes of 43, wherein said composition represents the treatment window bigger than being administered hydroxyurea.

48. such as claim 42 or the purposes of 43, wherein said composition represents the treatment window bigger than being administered sodium butyrate.